ALK4:ActRIIB heteromultimers and uses thereof

ABSTRACT

In certain aspects, the disclosure provides soluble heteromeric polypeptide complexes comprising an extracellular domain of an ALK4 receptor and an extracellular domain of ActRIIB. In certain aspects, such soluble ALK4:ActRIIB complexes may be used to regulate (promote or inhibit) growth of tissues or cells including, for example, muscle, bone, cartilage, fat, neural tissue, tumors, and/or cancerous cells. In certain aspects, such ALK4:ActRIIB complexes are can be used to improve muscle formation, bone formation, metabolic parameters, and disorders associated with these tissues, cellular networks, kidney, and endocrine systems.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. provisionalapplication Ser. No. 62/143,579, filed Apr. 6, 2015; and U.S.provisional application Ser. No. 62/220,836, filed Sep. 18, 2015. Thedisclosures of each of the foregoing applications are herebyincorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 25, 2016, isnamed PHPH109101_SL.txt and is 122,692 bytes in size.

BACKGROUND OF THE INVENTION

The transforming growth factor-beta (TGF-beta) superfamily contains avariety of growth factors that share common sequence elements andstructural motifs. These proteins are known to exert biological effectson a large variety of cell types in both vertebrates and invertebrates.Members of the superfamily perform important functions during embryonicdevelopment in pattern formation and tissue specification and caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis,neurogenesis, and epithelial cell differentiation. The family is dividedinto two general phylogenetic clades: the more recently evolved membersof the superfamily, which includes TGF-betas, activins, and nodal andthe clade of more distantly related proteins of the superfamily, whichincludes a number of BMPs and GDFs [Hinck (2012) FEBS Letters586:1860-1870]. TGF-beta family members have diverse, oftencomplementary biological effects. By manipulating the activity of amember of the TGF-beta family, it is often possible to cause significantphysiological changes in an organism. For example, the Piedmontese andBelgian Blue cattle breeds carry a loss-of-function mutation in the GDF8(also called myostatin) gene that causes a marked increase in musclemass [Grobet et al. (1997) Nat Genet 17(1):71-4]. Furthermore, inhumans, inactive alleles of GDF8 are associated with increased musclemass and, reportedly, exceptional strength [Schuelke et al. (2004) NEngl J Med 350:2682-8].

Changes in fibrosis, muscle, bone, fat, red blood cells, and othertissues may be achieved by enhancing or inhibiting intracellularsignaling (e.g., SMAD 1, 2, 3, 5, and/or 8) that is mediated by ligandsof the TGF-beta family. Thus, there is a need for agents that regulatethe activity of various ligands of the TGF-beta superfamily.

SUMMARY OF THE INVENTION

As described herein, it has been discovered that an ALK4:ActRIIBheterodimer protein complex is a unique antagonist of ligands of theTGF-beta superfamily, exhibiting a different ligand-bindingprofile/selectivity compared to corresponding ActRIIB and ALK4homodimers. In particular, an exemplary ALK4:ActRIIB heterodimerdisplays enhanced binding to activin B compared to either homodimer,retains strong binding to activin A, GDF8, and GDF11 as observed withActRIIB homodimer, and exhibits substantially reduced binding to BMP9,BMP10, and GDF3. In fact, the ALK4:ActRIIB heterodimer displays low tono observable affinity for BMP9, whereas this ligand binds strongly toActRIIB homodimer. See FIG. 6. These results therefore demonstrate thatALK4:ActRIIB heterodimers are a more selective antagonists (inhibitors)of certain ligands of the TGF-beta superfamily compared to ActRIIBhomodimers. Accordingly, an ALK4:ActRIIB heterodimer will be more usefulthan an ActRIIB homodimer in certain applications where such selectiveantagonism is advantageous. Examples include therapeutic applicationswhere it is desirable to antagonize one or more of activin (e.g.,activin A, activin B, activin AB, activin AC), GDF8, and GDF11 withdecreased antagonism of one or more of BMP9, BMP10, and GDF3.

Moreover, ALK4:ActRIIB heterodimer produced certain biological effectsstrikingly similar to those of an ActRIIB homodimer despite differentialligand selectivity of the two complexes. For example, ALK4:ActRIIBheterodimer exerts beneficial anabolic effects on skeletal muscle andbone as well as catabolic effects on adipose tissue, very similar tothose of an ActRIIB-Fc homodimer. However, unlike ActRIIB homodimer,ActRIIB:ALK4 heterodimer exhibits only low-affinity or transient bindingto BMP9 and BMP10 and so should have little to no concurrent inhibitionon processes mediated by those ligands, such as angiogenesis. This novelselectivity may be useful, for example, in treating patients in need ofstimulatory effects on muscle and bone, and/or inhibitory effects onfat, but not in need of altered angiogenesis. In addition, ALK4:ActRIIBheterodimer had various beneficial effects in a mouse model of kidneydisease, particularly on treating or preventing kidney damage,inflammation, and fibrosis. Therefore, while not wishing to be bound toa particular mechanisms of action, it is expected that ALK4:ActRIIBheteromultimers, as well as variants thereof, that bind to/inhibit atleast one or more of activin (e.g., activin A, activin B, activin AB,and activin AC), GDF8, and/or GDF11 will be useful agents for promotingbeneficial anabolic effects on skeletal muscle and bone, cataboliceffects on adipose tissue, and beneficial effects on kidney disease.Furthermore, it is expected that other antagonists (inhibitors), orcombinations of antagonists, that mimic the binding/inhibitoryproperties of the ALK4:ActRIIB heterodimers described herein as well asagents that directly or indirectly antagonize ALK4 and/or ActRIIBreceptors, agents that directly or indirectly antagonize ALK4- and/orActRIIB-binding ligands, agents that directly or indirectly antagonizedownstream signaling mediators (e.g., Smads), and/or agents thatdirectly or indirectly antagonize TGF-beta superfamily co-receptors willhave similar biological effects in vivo including, for example,stimulatory effects on muscle and bone and inhibitory effects on fat.These antagonistic mimetic are collectively referred to herein as“ALK4:ActRIIB antagonists” or “ALK4:ActRIIB inhibitors”.

Therefore, the present disclosure provides, in part, heteromultimercomplexes (heteromultimers) comprising at least one ALK4 polypeptide andat least one ActRIIB polypeptide (ALK4:ActRIIB heteromultimers).Preferably, ALK4 polypeptides comprise a ligand-binding domain of anALK4 receptor, for example, a portion of the ALK4 extracellular domain.Similarly, ActRIIB polypeptides generally comprise a ligand-bindingdomain of an ActRIIB receptor, for example, a portion of the ActRIIBextracellular domain. Preferably, such ALK4 and ActRIIB polypeptides, aswell as resultant heteromultimers thereof, are soluble.

In certain aspects, an ALK4:ActRIIB heteromultimer comprises an ALK4amino acid sequence that is at least 70% identical to a polypeptide thatbegins at any one of amino acids 24-34 of SEQ ID NO: 9 (e.g., aminoacids 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34) and ends at anyone of amino acids 101-126 of SEQ ID NO: 9 (e.g., amino acids 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, and 126). For example,ALK4:ActRIIB heteromultimers may comprise an amino acid sequence that isat least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 34-101 ofSEQ ID NO: 9. In some embodiments, ALK4:ActRIIB heteromultimers maycomprise an amino acid sequence that is at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to amino acids 24-126 of SEQ ID NO: 9. In otherembodiments, ALK4:ActRIIB heteromultimers may comprise an ALK4 aminoacid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 10. In still other embodiments, ALK4:ActRIIB heteromultimersmay comprise an ALK4 amino acid sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 20.

In certain aspects, an ALK4:ActRIIB heteromultimer comprises an ActRIIBamino acid sequence that is at least 70% identical to a polypeptide thatbegins at any one of amino acids 20-29 of SEQ ID NO: 1 (e.g., aminoacids 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29) and ends at any one ofamino acids 109-134 (109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, and 134) of SEQ ID NO: 1. For example, ALK4:ActRIIB heteromultimersmay comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to amino acids 29-109 of SEQ ID NO: 1. In someembodiments, ALK4:ActRIIB heteromultimers may comprise an amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to aminoacids 25-131 of SEQ ID NO: 1. In other embodiments, ALK4:ActRIIBheteromultimers may comprise an ActRIIB amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. In stillother embodiments, ALK4:ActRIIB heteromultimers may comprise an ActRIIBamino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 3. In even other embodiments, ALK4:ActRIIB heteromultimersmay comprise an ActRIIB amino acid sequence that is at least 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 5. In still even otherembodiments, ALK4:ActRIIB heteromultimers may comprise an ActRIIB aminoacid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 6. In certain preferred embodiments, ALK4:ActRIIBheteromultimers do not comprise an ActRIIB polypeptide comprising anacidic amino acid (e.g., the naturally occurring amino acids E or D oran artificial acidic amino acid) at the position corresponding to L79 ofSEQ ID NO: 1.

Various combinations of the ALK4 and ActRIIB polypeptides describedherein are also contemplated with respect to ALK4:ActRIIBheteromultimers. For example, in certain aspects, an ALK4:ActRIIBheteromultimer may comprise a) a polypeptide comprising, consistingessentially of, or consisting of an ALK4 amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 34-101 of SEQID NO: 9; and b) a polypeptide comprising, or consisting essentially of,or consisting of an ActRIIB amino acid sequence that is at least 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to amino acids 29-109 of SEQ ID NO: 1.In certain aspects, an ALK4:ActRIIB heteromultimer may comprise a) apolypeptide comprising, consisting essentially of, or consisting of anALK4 amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to amino acids 24-126 of SEQ ID NO: 9; and b) a polypeptidecomprising, or consisting essentially of, or consisting of an ActRIIBamino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto amino acids 25-131 of SEQ ID NO: 1. In other aspects, an ALK4:ActRIIBheteromultimer may comprise a) a polypeptide comprising, consistingessentially of, or consisting of an ALK4 amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10; and b) apolypeptide comprising, or consisting essentially of, or consisting ofan ActRIIB amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 2. In other aspects, an ALK4:ActRIIBheteromultimer may comprise a) a polypeptide comprising, consistingessentially of, or consisting of an ALK4 amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20; and b) apolypeptide comprising, or consisting essentially of, or consisting ofan ActRIIB amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 2. In even other aspects, an ALK4:ActRIIBheteromultimer may comprise a) a polypeptide comprising, consistingessentially of, or consisting of an ALK4 amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10; and b) apolypeptide comprising, or consisting essentially of, or consisting ofan ActRIIB amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 3. In even other aspects, an ALK4:ActRIIBheteromultimer may comprise a) a polypeptide comprising, consistingessentially of, or consisting of an ALK4 amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20; and b) apolypeptide comprising, or consisting essentially of, or consisting ofan ActRIIB amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 3. In still other aspects, an ALK4:ActRIIBheteromultimer may comprise a) a polypeptide comprising, consistingessentially of, or consisting of an ALK4 amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10; and b) apolypeptide comprising, or consisting essentially of, or consisting ofan ActRIIB amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 5. In still other aspects, an ALK4:ActRIIBheteromultimer may comprise a) a polypeptide comprising, consistingessentially of, or consisting of an ALK4 amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20; and b) apolypeptide comprising, or consisting essentially of, or consisting ofan ActRIIB amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 5. In still even other aspects, an ALK4:ActRIIBheteromultimer may comprise a) a polypeptide comprising, consistingessentially of, or consisting of an ALK4 amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10; and b) apolypeptide comprising, or consisting essentially of, or consisting ofan ActRIIB amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 6. In still even other aspects, an ALK4:ActRIIBheteromultimer may comprise a) a polypeptide comprising, consistingessentially of, or consisting of an ALK4 amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20; and b) apolypeptide comprising, or consisting essentially of, or consisting ofan ActRIIB amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 6.

As described herein, ALK4:ActRIIB heteromultimer structures include, forexample, heterodimers, heterotrimers, heterotetramers, heteropentamers,and higher order heteromultimer complexes. See, e.g., FIGS. 1, 2, and8-10. In certain preferred embodiments, ALK4:ActRIIB heteromultimers areheterodimers.

In certain aspects, ALK4 and/or ActRIIB polypeptides may be fusionproteins. For example, in some embodiments, an ALK4 polypeptide may be afusion protein comprising an ALK4 polypeptide domain and one or moreheterologous (non-ALK4) polypeptide domains. Similarly, in someembodiments, an ActRIIB polypeptide may be a fusion protein comprisingan ActRIIB polypeptide domain and one or more heterologous (non-ActRIIB)polypeptide domains.

Optionally, ALK4 polypeptides are connected directly (fused) to one ormore heterologous domains, or an intervening sequence, such as a linker,may be positioned between the amino acid sequence of the ALK4polypeptide and the one or more heterologous domains. Similarly, theActRIIB polypeptide may be connected directly (fused) to one or moreheterologous domains, or an intervening sequence, such as a linker, maybe positioned between the amino acid sequence of the ActRIIB polypeptideand the one or more heterologous domains. Linkers may correspond to theroughly 15 amino acid unstructured region at the C-terminal end of theextracellular domain of ActRIIB or ALK4 (the “tail”), or it may be anartificial sequence of between 5 and 15, 20, 30, 50, 100 or more aminoacids that are relatively free of secondary structure. A linker may berich in glycine and proline residues and may, for example, containrepeating sequences of threonine/serine and glycines. Examples oflinkers include, but are not limited to, the sequences TGGG (SEQ ID NO:17), SGGG (SEQ ID NO: 18), TGGGG (SEQ ID NO: 15), SGGGG (SEQ ID NO: 16),GGGGS (SEQ ID NO: 58), GGGG (SEQ ID NO: 14), and GGG (SEQ ID NO: 13). Insome embodiments, the one or more heterologous domains that provide adesirable property to the ALK4 and/or ActRIIB fusion proteins including,for example, improved pharmacokinetics, easier purification, targetingto particular tissues, etc. For example, a heterologous domain of afusion protein may enhance one or more of in vivo stability, in vivohalf-life, uptake/administration, tissue localization or distribution,formation of protein complexes, multimerization of the fusion protein,and/or purification. An ALK4 or ActRIIB fusion protein may include animmunoglobulin Fc domain (wild-type or mutant) or a serum albumin. Insome embodiments, ALK4 and/or ActRIIB polypeptides may comprise apurification subsequence, such as an epitope tag, a FLAG tag, apolyhistidine sequence, and a GST fusion.

In certain embodiments, ALK4:ActRIIB heteromultimers described hereincomprise an ALK4 polypeptide covalently, or non-covalently, associatedwith an ActRIIB polypeptide wherein the ALK4 polypeptide comprises anALK4 domain and an amino acid sequence of a first member (or secondmember) of an interaction pair and the ActRIIB polypeptide comprises anActRIIB polypeptide and an amino acid sequence of a second member (orfirst member) of the interaction pair. Interaction pairs describedherein are designed to promote dimerization or form higher ordermultimers. See, e.g., FIGS. 1, 2, and 8-10 In some embodiments, theinteraction pair may be any two polypeptide sequences that interact toform a complex, particularly a heterodimeric complex although operativeembodiments may also employ an interaction pair that forms a homodimericsequence. The first and second members of the interaction pair may be anasymmetric pair, meaning that the members of the pair preferentiallyassociate with each other rather than self-associate (i.e., guidedinteraction pairs). Accordingly, first and second members of anasymmetric interaction pair may associate to form a heterodimericcomplex. Alternatively, the interaction pair may be unguided, meaningthat the members of the pair may associate with each other orself-associate without substantial preference and thus may have the sameor different amino acid sequences. Accordingly, first and second membersof an unguided interaction pair may associate to form a homodimercomplex or a heterodimeric complex. Optionally, the first member of theinteraction action pair (e.g., an asymmetric pair or an unguidedinteraction pair) associates covalently with the second member of theinteraction pair. Optionally, the first member of the interaction actionpair (e.g., an asymmetric pair or an unguided interaction pair)associates non-covalently with the second member of the interactionpair. Optionally, the first member of the interaction action pair (e.g.,an asymmetric pair or an unguided interaction pair) associates throughboth covalent and non-covalent mechanisms with the second member of theinteraction pair.

In some embodiments, ALK4 polypeptides are fusion proteins that comprisean Fc domain of an immunoglobulin. Similarly, in some embodiments,ActRIIB polypeptides are fusion proteins that comprise an Fc domain ofan immunoglobulin. Traditional Fc fusion proteins and antibodies areexamples of unguided interaction pairs, whereas a variety of engineeredFc domains have been designed as asymmetric interaction pairs [Spiess etal (2015) Molecular Immunology 67(2A): 95-106]. Therefore, a firstmember and/or a second member of an interaction pair described hereinmay comprise a constant domain of an immunoglobulin, including, forexample, the Fc portion of an immunoglobulin. For example, a firstmember of an interaction pair may comprise an amino acid sequence thatis derived from an Fc domain of an IgG (IgG1, IgG2, IgG3, or IgG4), IgA(IgA1 or IgA2), IgE, or IgM immunoglobulin. Such immunoglobulin domainsmay comprise one or more amino acid modifications (e.g., deletions,additions, and/or substitutions) that promote ALK4:ActRIIBheteromultimer formation. For example, the first member of aninteraction pair may comprise, consist essentially of, or consist of anamino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ IDNOs: 23-37. Similarly, a second member of an interaction pair maycomprise an amino acid sequence that is derived from an Fc domain of anIgG (IgG1, IgG2, IgG3, or IgG4), IgA (IgA1 or IgA2), IgE, or IgM. Suchimmunoglobulin domains may comprise one or more amino acid modifications(e.g., deletions, additions, and/or substitutions) that promoteALK4:ActRIIB heteromultimer formation. For example, the second member ofan interaction pair may comprise, consist essentially of, or consist ofan amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one ofSEQ ID NOs: 23-37. In some embodiments, a first member and a secondmember of an interaction pair comprise Fc domains derived from the sameimmunoglobulin class and subtype. In other embodiments, a first memberand a second member of an interaction pair comprise Fc domains derivedfrom different immunoglobulin classes or subtypes. In some embodiments,an ALK4:ActRIIB heterodimer comprises i) an ALK4 polypeptide comprising,consisting essentially of, or consisting of an amino acid sequence thatis at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44, andii) an ActRIIB polypeptide comprising, consisting essentially of, orconsisting of an amino acid sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 41. In other embodiments, anALK4:ActRIIB heterodimer comprises i) an ALK4 polypeptide comprising,consisting essentially of, or consisting of an amino acid sequence thatis at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 48, andii) an ActRIIB polypeptide comprising, consisting essentially of, orconsisting of an amino acid sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 46.

Optionally, a first member and/or a second member of an interaction pair(e.g., an asymmetric pair or an unguided interaction pair) comprise amodified constant domain of an immunoglobulin, including, for example, amodified Fc portion of an immunoglobulin. For example, protein complexesof the disclosure may comprise a first modified Fc portion of an IgGcomprising, consisting essentially of, or consisting of an amino acidsequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to an amino acid sequence selected from thegroup: SEQ ID NOs: 23-37 and a second modified Fc portion of an IgG,which may be the same or different from the amino acid sequence of thefirst modified Fc portion of the IgG, comprising, consisting essentiallyof, or consisting of an amino acid sequence that is at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to anamino acid sequence selected from the group: SEQ ID NOs: 23-37. In someembodiments, ALK4:ActRIIB heteromultimers comprise: a) an ALK4 (orActRIIB) fusion protein comprising an immunoglobulin domain thatcomprises, consisting essentially of, or consisting of an amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO: 23, optionally wherein the immunoglobulin domain comprises apositively charged amino acid (e.g., K, R, or H) at the positionscorresponding to residues 134 and 177 of SEQ ID NO: 23, and furtheroptionally wherein the immunoglobulin domain does not comprise apositively charged amino acid (e.g., K, R, or H) at the positioncorresponding to residue 225 of SEQ ID NO: 23, and b) an ActRIIB (orALK4) fusion protein comprising an immunoglobulin domain that comprises,consisting essentially of, or consisting of an amino acid sequence thatis at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 24,optionally wherein the immunoglobulin domain comprises a negativelycharged (e.g., D or E) amino acid at the positions corresponding toresidues 170 and 187 of SEQ ID NO: 24, and further optionally whereinthe immunoglobulin domain comprises a positively charged amino acid(e.g., K, R, or H) at the position corresponding to residue 225 of SEQID NO: 24. In other embodiments, ALK4:ActRIIB heteromultimers comprise:a) an ALK4 (or ActRIIB) fusion protein comprising an immunoglobulindomain that comprises, consisting essentially of, or consisting of anamino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 27, optionally wherein the immunoglobulin domain comprisesa C at the position corresponding to residue 132 of SEQ ID NO: 27 and aW at the position corresponding to residue 144 of SEQ ID NO: 27, andfurther optionally wherein the immunoglobulin domain does not comprise apositively charged amino acid (e.g., K, R, or H) at the positioncorresponding to residue 225 of SEQ ID NO: 27, and b) an ActRIIB (orALK4) fusion protein comprising an immunoglobulin domain that comprises,consisting essentially of, or consisting of an amino acid sequence thatis at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28,optionally wherein the immunoglobulin domain comprises a S at theposition corresponding to residue 144 of SEQ ID NO: 28, an A at theposition corresponding to residue 146 of SEQ ID NO: 28, and a V at theposition corresponding to residue 185 of SEQ ID NO: 28, and furtheroptionally wherein the immunoglobulin domain does not comprises apositively charged amino acid (e.g., K, R, or H) at the positioncorresponding to residue 225 of SEQ ID NO: 28.

In certain aspects, an ALK4:ActRIIB heteromultimer comprises, consistsessentially of, or consists of an ActRIIB amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 39. In someembodiments, an ALK4:ActRIIB heteromultimer comprises, consistsessentially of, or consists of an ActRIIB amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 41. In certainaspects, an ALK4:ActRIIB heteromultimer comprises, consists essentiallyof, or consists of an ALK4 amino acid sequence that is at least 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to SEQ ID NO: 42. In some embodiments,an ALK4:ActRIIB heteromultimer comprises, consists essentially of, orconsists of an ALK4 amino acid sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 44. Various combinations of theALK4 and ActRIIB fusion polypeptides described herein are alsocontemplated with respect to ALK4:ActRIIB heteromultimers. For example,in some embodiments, an ALK4:ActRIIB heteromultimer may comprise a) apolypeptide comprising, consisting essentially of, or consisting of anALK4 amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 44; and b) a polypeptide comprising, orconsisting essentially of, or consisting of an ActRIIB amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO: 41. In some embodiments, an ALK4:ActRIIB heteromultimer may comprisea) a polypeptide comprising, consisting essentially of, or consisting ofan ALK4 amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 48; and b) a polypeptide comprising, orconsisting essentially of, or consisting of an ActRIIB amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO: 46.

Optionally, an ALK4 and/or ActRIIB polypeptide comprises one or moremodified amino acid residues selected from: a glycosylated amino acid, aPEGylated amino acid, a farnesylated amino acid, an acetylated aminoacid, a biotinylated amino acid, an amino acid conjugated to a lipidmoiety, and an amino acid conjugated to an organic derivatizing agent.ALK4 and/or ActRIIB polypeptides may comprise at least one N-linkedsugar, and may include two, three or more N-linked sugars. Suchpolypeptides may also comprise O-linked sugars. ALK4 and/or ActRIIBpolypeptides may be produced in a variety of cell lines that glycosylatethe protein in a manner that is suitable for patient use, includingengineered insect or yeast cells, and mammalian cells such as COS cells,CHO cells, HEK cells and NSO cells. In some embodiments an ALK4 and/orActRIIB polypeptide is glycosylated and has a glycosylation patternobtainable from a Chinese hamster ovary cell line. PreferablyALK4:ActRIIB heteromultimer complexes of the disclosure exhibit a serumhalf-life of at least 4, 6, 12, 24, 36, 48, or 72 hours in a mammal(e.g., a mouse or a human). Optionally, ALK4:ActRIIB heteromultimers mayexhibit a serum half-life of at least 6, 8, 10, 12, 14, 20, 25, or 30days in a mammal (e.g., a mouse or a human).

In certain aspects, ALK4:ActRIIB heteromultimers of the disclosure bindto one or more TGF-beta superfamily ligands. Optionally, ALK4:ActRIIBheteromultimers bind to one or more of these ligands with a K_(D) ofless than or equal to 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or 10⁻¹²M. For example,in some embodiments, ALK4:ActRIIB heteromultimers bind to activin B. Insome embodiments, ALK4:ActRIIB heteromultimers bind to activin A. Insome embodiments, ALK4:ActRIIB heteromultimers bind to activin AB. Insome embodiments, ALK4:ActRIIB heteromultimers bind to activin C. Insome embodiments, ALK4:ActRIIB heteromultimers bind to activin AC. Insome embodiments, ALK4:ActRIIB heteromultimers bind to activin BC. Insome embodiments, ALK4:ActRIIB heteromultimers bind to activin BC. Insome embodiments, ALK4:ActRIIB heteromultimers bind to activin BE. Insome embodiments, ALK4:ActRIIB heteromultimers bind to GDF11. In someembodiments, ALK4:ActRIIB heteromultimers bind to GDF8. In someembodiments, ALK4:ActRIIB heteromultimers bind to BMP6. In someembodiments, ALK4:ActRIIB heteromultimers bind to GDF3. In someembodiments, ALK4:ActRIIB heteromultimers bind to BMP10. In someembodiments, ALK4:ActRIIB heteromultimers do not bind to, or do notsubstantially bind to, BMP9. In some embodiments, ALK4:ActRIIBheteromultimers bind to activin B with stronger affinity compared to acorresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIBheteromultimers bind to GDF3 with weaker affinity compared to acorresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIBheteromultimers bind to BMP10 with weaker affinity compared to acorresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIBheteromultimers bind to BMP9 with weaker affinity compared to acorresponding ActRIIB homomultimer.

In general, ALK4:ActRIIB heteromultimers of the disclosure antagonize(inhibit) one or more activities of at least one TGF-beta superfamilyligand, and such alterations in activity may be measured using variousassays known in the art, including, for example, a cell-based assay suchas those described herein. In certain aspects, ALK4:ActRIIBheteromultimers may be used to inhibit signaling (e.g., Smad 2/3 and/orSmad 1/5/8 signaling) mediated by one or more TGFβ superfamily ligandsin, for example, a cell-based assay. For example, in some embodiments,ALK4:ActRIIB heteromultimers inhibit activin signaling in a cell-basedassay. In some embodiments, ALK4:ActRIIB heteromultimers inhibit activinsignaling in a cell-based assay. In some embodiments, ALK4:ActRIIBheteromultimers inhibit activin A signaling in a cell-based assay. Insome embodiments, ALK4:ActRIIB heteromultimers inhibit activin Bsignaling in a cell-based assay. In some embodiments, ALK4:ActRIIBheteromultimers inhibit activin AB signaling in a cell-based assay. Insome embodiments, ALK4:ActRIIB heteromultimers inhibit activin Csignaling in a cell-based assay. In some embodiments, ALK4:ActRIIBheteromultimers inhibit activin AC signaling in a cell-based assay. Insome embodiments, ALK4:ActRIIB heteromultimers inhibit activin BCsignaling in a cell-based assay. In some embodiments, ALK4:ActRIIBheteromultimers inhibit activin E signaling in a cell-based assay. Insome embodiments, ALK4:ActRIIB heteromultimers inhibit activin AEsignaling in a cell-based assay. In some embodiments, ALK4:ActRIIBheteromultimers inhibit activin CE signaling in a cell-based assay. Insome embodiments, ALK4:ActRIIB heteromultimers inhibit GDF11 signalingin a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimersinhibit GDF8 signaling in a cell-based assay. In some embodiments,ALK4:ActRIIB heteromultimers inhibit BMP6 signaling in a cell-basedassay. In some embodiments, ALK4:ActRIIB heteromultimers inhibit GDF3signaling in a cell-based assay. In some embodiments, ALK4:ActRIIBheteromultimers inhibit BMP10 signaling in a cell-based assay. In someembodiments, ALK4:ActRIIB heteromultimers does not inhibit, or does notsubstantially inhibit, BMP9 signaling in a cell-based assay. In someembodiments, ALK4:ActRIIB heteromultimers are stronger inhibitors ofactivin B signaling in a cell-based assay. In some embodiments,ALK4:ActRIIB heteromultimers are weaker inhibitors of GDF3 signaling ina cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimersare weaker inhibitors of BMP10 signaling in a cell-based assay. In someembodiments, ALK4:ActRIIB heteromultimers are weaker inhibitors of BMP9signaling in a cell-based assay.

Any of the ALK4:ActRIIB heteromultimers as well as ALK4:ActRIIBantagonists described herein may be formulated as a pharmaceuticalpreparation (compositions). In some embodiments, pharmaceuticalpreparations comprise a pharmaceutically acceptable carrier. Apharmaceutical preparation will preferably be pyrogen-free (meaningpyrogen free to the extent required by regulations governing the qualityof products for therapeutic use). A pharmaceutical preparation may alsoinclude one or more additional compounds such as a compound that is usedto treat a disorder/condition described herein. In general, ALK4:ActRIIBheteromultimer pharmaceutical preparations are substantially free ofALK4 and/or ActRIIB homomultimers. For example, in some embodiments,ALK4:ActRIIB heteromultimer pharmaceutical preparations comprise lessthan about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1%ALK4 homomultimers. In some embodiments, ALK4:ActRIIB heteromultimerpharmaceutical preparations comprise less than about 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or less than about 1% ActRIIB homomultimers.

In certain aspects, the disclosure provides nucleic acids encoding anALK4 or ActRIIB polypeptide as described herein. For example, an ActRIIBnucleic acid may comprise, consists essentially of, or consists of anucleic acid that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical tothe sequence of 73-396 of SEQ ID NO: 7 or one that hybridizes understringent conditions to the complement of nucleotides 73-396 of SEQ IDNO: 7. Such an nucleic acid may be one that comprises the sequence ofSEQ ID NOs: 8 or 40. In some embodiments, an ActRIIB nucleic acidscomprises, consists essentially of, or consists of a nucleotide sequencethat is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQID Nos: 7, 8, and 40. Similarly, an ALK4 nucleic acid may comprise,consists essentially of, or consists of a nucleic acid that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to the sequence of 70-378 of SEQID NO: 11 or one that hybridizes under stringent conditions to thecomplement of nucleotides 70-378 of SEQ ID NO: 11. Such an ALK4 nucleicacid may be one that comprises the sequence of SEQ ID NOs: 12, 22, or43. In some embodiments, an ALK4 nucleic acids comprises, consistsessentially of, or consists of a nucleotide sequence that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 11, 12,21, 22, and 43.

In certain aspects, the present disclosure provides nucleic acidssequence comprising a coding sequence for and ALK4 polypeptide and acoding sequence for the ActRIIB polypeptide. For example, in someembodiments, nucleic acids of the disclosure a) comprises, consistsessentially of, or consists of a nucleotide sequence that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 11, 12,21, 22, and 43, and b) comprises, consists essentially of, or consistsof a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to any one of SEQ ID Nos: 7, 8, and 40. Preferably, ALK4and/or ActRIIB nucleic acids are isolated and/or recombinant nucleicacids. Nucleic acids disclosed herein may be operably linked to apromoter for expression. The present disclosure further provides vectorscomprising such ALK4 and/or ActRIIB polynucleotides as well as cells(e.g., CHO cells), preferably cells isolated from a human or othervertebrate species, comprising such ALK4 and/or ActRIIB polynucleotidesas well as vectors comprising such ALK4 and/or ActRIIB polynucleotides.

In certain aspects, an ALK4 polypeptides and/or ActRIIB polypeptides maybe expressed in a mammalian cell line, optionally a cell line thatmediates suitably natural glycosylation of the ActRIIB or ALK4 proteinso as to diminish the likelihood of an unfavorable immune response in apatient (including the possibility of veterinary patients). Human andCHO cell lines have been used successfully, and it is expected thatother common mammalian expression vectors will be useful. Thus thedisclosure provides cultured cells comprising any of the nucleic acidsdisclosed herein. Such cells may be mammalian cells, including CHOcells, NSO cells, HEK cells and COS cells. Other cells may be chosendepending on the species of the intended patient. Other cells aredisclosed herein. Cultured cells are understood to mean cells maintainedin laboratory or other man-made conditions (e.g., frozen, or in media)and not part of a living organism.

In certain aspects, the disclosure provides methods for making any ofthe ALK4 and ActRIIB polypeptides described herein as well asALK4:ActRIIB heteromultimer complexes comprising such polypeptides. Sucha method may include expressing any of the nucleic acids disclosedherein in a suitable cell (e.g., CHO cell or a COS cell). For example,in some embodiments a method of making a heteromultimer comprising anALK4 polypeptide and an ActRIIB polypeptide comprises: a) culturing acell under conditions suitable for expression of an ALK4 polypeptide andan ActRIIB polypeptide, wherein the cell comprises an ALK4polynucleotide and an ActRIIB polynucleotide; and b) recovering theheteromultimer so expressed. Alternatively, a method of making aheteromultimer comprising an ALK4 polypeptide and an ActRIIB polypeptidemay comprise: a) culturing a first cell under conditions suitable forexpression of an ALK4 polypeptide, wherein the first cell comprises anALK4 polynucleotide; b) recovering the ALK4 polypeptide so expressed; c)culturing a second cell under conditions suitable for expression of anActRIIB polypeptide, wherein the second cell comprises an ActRIIBpolynucleotide; d) recovering the ActRIIB polypeptide so expressed; e)combining the recovered ALK4 polypeptide and the recovered ActRIIBpolypeptide under conditions suitable for ALK4:ActRIIB heteromultimerformation; and f) recovering the ALK4:ActRIIB heteromultimer. In certainembodiments, ALK4 and/or ActRIIB polypeptides are expressed using a TPAleader sequence (e.g., SEQ ID NO: 38). In certain embodiments, ALK4and/or ActRIIB polypeptides are expressed in a CHO cell. ALK4 andActRIIB polypeptides described herein, as well as protein complexes ofthe same, may be recovered as crude, partially purified, or highlypurified fractions using any of the well-known techniques for obtainingprotein from cell cultures. In general, such methods result inALK4:ActRIIB heteromultimers that substantially free of ALK4 and/orActRIIB homomultimers. For example, in some embodiments, methods forproducing ALK4:ActRIIB heteromultimers result in less than about 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% ALK4homomultimers. In some embodiments, methods for producing ALK4:ActRIIBheteromultimers result in less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%,3%, 2%, or less than about 1% ActRIIB homomultimers. In someembodiments, methods for producing ALK4:ActRIIB heteromultimers resultin less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less thanabout 1% ALK4 homomultimers and less than about 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, or less than about 1% ActRIIB homomultimers.

The disclosure further provides methods and ALK4:ActRIIB antagonists(e.g., ALK4:ActRIIB heteromultimers) for use in the treatment orprevention of various ALK4:ActRIIB-associated diseases and conditionsassociated with, for example, muscle, bone, fat, red blood cells, andother tissues. Such disease and disorders include, but are not limitedto, disorders associated with muscle loss or insufficient muscle growth(e.g., muscle atrophy; muscular dystrophy, including Duchenne musculardystrophy, Becker muscular dystrophy, and facioscapulohumeral musculardystrophy; amyotrophic lateral sclerosis; and cachexia) and disordersassociated with undesirable weight gain (e.g., obesity, type 2 diabetesor non-insulin dependent diabetes mellitus (NIDDM), cardiovasculardisease, hypertension, osteoarthritis, stroke, respiratory problems, andgall bladder disease). In some embodiments, ALK4:ActRIIB antagonists(e.g., heteromultimers) may be used to decrease body fat content orreduce the rate of increase in body fat content in a subject in needthereof. In some embodiments, ALK4:ActRIIB antagonists (e.g.,heteromultimers) may be used to reduce cholesterol and/or triglyceridelevels in a patient. In some embodiments, ALK4:ActRIIB antagonists(e.g., heteromultimers) may be used to treat or prevent fibrosis or afibrosis-associated disorder or condition (e.g., renal failure, chronicrenal disease, cystic fibrosis, and myelofibrosis).

The disclosure further provides methods and ALK4:ActRIIB antagonists foruse in the treatment or prevention of various ALK4:ActRIIB—associateddiseases and conditions associated with, for example, the kidney. Suchdiseases or conditions include, for example, chronic kidney disease orfailure, acute kidney disease or failure, patients that have stage 1kidney disease, patients that have stage 2 kidney disease, patients thathave stage 3 kidney disease, patients that have stage 4 kidney disease,patients that have stage 5 kidney disease, non-diabetic kidney diseases,glomerulonephritis, interstitial nephritis, diabetic kidney diseases,diabetic nephropathy, glomerulosclerosis, rapid progressiveglomerulonephritis, renal fibrosis, Alport syndrome, IDDM nephritis,mesangial proliferative glomerulonephritis, membranoproliferativeglomerulonephritis, crescentic glomerulonephritis, renal interstitialfibrosis, focal segmental glomerulosclerosis, membranous nephropathy,minimal change disease, pauci-immune rapid progressiveglomerulonephritis, IgA nephropathy, polycystic kidney disease, Dent'sdisease, nephrocytinosis, Heymann nephritis, autosomal dominant (adult)polycystic kidney disease, autosomal recessive (childhood) polycystickidney disease, acute kidney injury, nephrotic syndrome, renal ischemia,podocyte diseases or disorders, proteinuria, glomerular diseases,membranous glomerulonephritis, focal segmental glomerulonephritis,pre-eclampsia, eclampsia, kidney lesions, collagen vascular diseases,benign orthostatic (postural) proteinuria, IgM nephropathy, membranousnephropathy, sarcoidosis, diabetes mellitus, kidney damage due to drugs,Fabry's disease, aminoaciduria, Fanconi syndrome, hypertensivenephrosclerosis, interstitial nephritis, Sickle cell disease,hemoglobinuria, myoglobinuria, Wegener's Granulomatosis, GlycogenStorage Disease Type 1, chronic kidney disease, chronic renal failure,low Glomerular Filtration Rate (GFR), nephroangiosclerosis, lupusnephritis, ANCA-positive pauci-immune crescentic glomerulonephritis,chronic allograft nephropathy, nephrotoxicity, renal toxicity, kidneynecrosis, kidney damage, glomerular and tubular injury, kidneydysfunction, nephritic syndrome, acute renal failure, chronic renalfailure, proximal tubal dysfunction, acute kidney transplant rejection,chronic kidney transplant rejection, non-IgA mesangioproliferativeglomerulonephritis, postinfectious glomerulonephritis, vasculitides withrenal involvement of any kind, any hereditary renal disease, anyinterstitial nephritis, renal transplant failure, kidney cancer, kidneydisease associated with other conditions (e.g., hypertension, diabetes,and autoimmune disease), Dent's disease, nephrocytinosis, Heymannnephritis, a primary kidney disease, a collapsing glomerulopathy, adense deposit disease, a cryoglobulinemia-associated glomerulonephritis,an Henoch-Schonlein disease, a postinfectious glomerulonephritis, abacterial endocarditis, a microscopic polyangitis, a Churg-Strausssyndrome, an anti-GBM-antibody mediated glomerulonephritis, amyloidosis,a monoclonal immunoglobulin deposition disease, a fibrillaryglomerulonephritis, an immunotactoid glomerulopathy, ischemic tubularinjury, a medication-induced tubulo-interstitial nephritis, a toxictubulo-interstitial nephritis, an infectious tubulo-interstitialnephritis, a bacterial pyelonephritis, a viral infectioustubulo-interstitial nephritis which results from a polyomavirusinfection or an HIV infection, a metabolic-induced tubulo-interstitialdisease, a mixed connective disease, a cast nephropathy, a crystalnephropathy which may results from urate or oxalate or drug-inducedcrystal deposition, an acute cellular tubulo-interstitial allograftrejection, a tumoral infiltrative disease which results from a lymphomaor a post-transplant lymphoproliferative disease, an obstructive diseaseof the kidney, vascular disease, a thrombotic microangiopathy, anephroangiosclerosis, an atheroembolic disease, a mixed connectivetissue disease, a polyarteritis nodosa, a calcineurin-inhibitorinduced-vascular disease, an acute cellular vascular allograftrejection, an acute humoral allograft rejection, early renal functiondecline (ERFD), end stage renal disease (ESRD), renal vein thrombosis,acute tubular necrosis, renal occlusion, acute interstitial nephritis,established chronic kidney disease, renal artery stenosis, ischemicnephropathy, uremia, drug and toxin-induced chronic tubulointerstitialnephritis, reflux nephropathy, kidney stones, Goodpasture's syndrome,normocytic normochromic anemia, renal anemia, diabetic chronic kidneydisease, IgG4-related disease, von Hippel-Lindau syndrome, tuberoussclerosis, nephronophthisis, medullary cystic kidney disease, renal cellcarcinoma, adenocarcinoma, nephroblastoma, lymphoma, leukemia,hyposialylation disorder, chronic cyclosporine nephropathy, renalreperfusion injury, renal dysplasia, azotemia, bilateral arterialocclusion, acute uric acid nephropathy, hypovolemia, acute bilateralobstructive uropathy, hypercalcemic nephropathy, hemolytic uremicsyndrome, acute urinary retention, malignant nephrosclerosis, postpartumglomerulosclerosis, scleroderma, non-Goodpasture's anti-GBM disease,microscopic polyarteritis nodosa, allergic granulomatosis, acuteradiation nephritis, post-streptococcal glomerulonephritis,Waldenstrom's macroglobulinemia, analgesic nephropathy, arteriovenousfistula, arteriovenous graft, dialysis, ectopic kidney, medullary spongekidney, renal osteodystrophy, solitary kidney, hydronephrosis,microalbuminuria, uremia, haematuria, hyperlipidemia, hypoalbuminaemia,lipiduria, acidosis, and hyperkalemia. In some embodiments, thedisclosure further provides methods and ALK4:ActRIIB antagonists for usein delaying or preventing progression from: stage 1 to stage 2 kidneydisease, stage 2 to stage 3 kidney disease, stage 3 to stage 4 kidneydisease, or stage 4 to stage 5 kidney disease. In some embodiments, thedisclosure further provides methods and ALK4:ActRIIB antagonists for usein preventing or reducing kidney inflammation. In some embodiments, thedisclosure further provides methods and ALK4:ActRIIB antagonists for usein preventing or reducing kidney damage. In some embodiments, thedisclosure further provides methods and ALK4:ActRIIB antagonists for usein preventing or reducing kidney fibrosis.

BRIEF DESCRIPTION OF THE DRAWINGS

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FIGS. 1A and 1B show two schematic examples of heteromeric proteincomplexes comprising type I receptor and type II receptor polypeptides.FIG. 1A depicts a heterodimeric protein complex comprising one type Ireceptor fusion polypeptide and one type II receptor fusion polypeptide,which can be assembled covalently or noncovalently via a multimerizationdomain contained within each polypeptide chain. Two assembledmultimerization domains constitute an interaction pair, which can beeither guided or unguided. FIG. 1B depicts a heterotetrameric proteincomplex comprising two heterodimeric complexes as depicted in FIG. 1A.Complexes of higher order can be envisioned.

FIG. 2 show a schematic example of a heteromeric protein complexcomprising a type I receptor polypeptide (indicated as “I”) (e.g. apolypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 97%, 98%, 99% or 100% identical to an extracellular domain ofan ALK4 protein from humans or other species such as those describedherein, e.g., SEQ ID Nos: 9, 10, 19, 20, 42, 44, 47, and 48) and a typeII receptor polypeptide (indicated as “II”) (e.g. a polypeptide that isat least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%or 100% identical to an extracellular domain of an ActRIIB protein fromhumans or other species as such as those described herein, e.g., SEQ IDNos: 1, 2, 3, 4, 5, 6, 39, 41, 45, and 46). In the illustratedembodiments, the type I receptor polypeptide is part of a fusionpolypeptide that comprises a first member of an interaction pair (“C₁”),and the type II receptor polypeptide is part of a fusion polypeptidethat comprises a second member of an interaction pair (“C₂”). In eachfusion polypeptide, a linker may be positioned between the type I ortype II receptor polypeptide and the corresponding member of theinteraction pair. The first and second members of the interaction pairmay be a guided (asymmetric) pair, meaning that the members of the pairassociate preferentially with each other rather than self-associate, orthe interaction pair may be unguided, meaning that the members of thepair may associate with each other or self-associate without substantialpreference and may have the same or different amino acid sequences.Traditional Fc fusion proteins and antibodies are examples of unguidedinteraction pairs, whereas a variety of engineered Fc domains have beendesigned as guided (asymmetric) interaction pairs [e.g., Spiess et al(2015) Molecular Immunology 67(2A): 95-106].

FIG. 3 shows an alignment of extracellular domains of human ActRIIA (SEQID NO: 49) and human ActRIIB (SEQ ID NO: 2) with the residues that arededuced herein, based on composite analysis of multiple ActRIIB andActRIIA crystal structures, to directly contact ligand indicated withboxes.

FIG. 4 shows a multiple sequence alignment of various vertebrate ActRIIBprecursor proteins without their intracellular domains (SEQ ID NOs:50-55, respectively) human ActRIIA precursor protein without itsintracellular domain (SEQ ID NO: 56), and a consensus ActRII precursorprotein (SEQ ID NO: 57).

FIG. 5 shows multiple sequence alignment of Fc domains from human IgGisotypes using Clustal 2.1. Hinge regions are indicated by dottedunderline. Double underline indicates examples of positions engineeredin IgG1 Fc to promote asymmetric chain pairing and the correspondingpositions with respect to other isotypes IgG2, IgG3 and IgG4. FIG. 5discloses SEQ ID NOS 31, 35, 32 and 33, respectively, in order ofappearance.

FIG. 6 shows comparative ligand binding data for an ALK4-Fc:ActRIIB-Fcheterodimeric protein complex compared to ActRIIB-Fc homodimer andALK4-Fc homodimer. For each protein complex, ligands are ranked byk_(off), a kinetic constant that correlates well with ligand signalinginhibition, and listed in descending order of binding affinity (ligandsbound most tightly are listed at the top). At left, yellow, red, green,and blue lines indicate magnitude of the off-rate constant. Solid blacklines indicate ligands whose binding to heterodimer is enhanced orunchanged compared with homodimer, whereas dashed red lines indicatesubstantially reduced binding compared with homodimer. As shown, theALK4-Fc:ActRIIB-Fc heterodimer displays enhanced binding to activin Bcompared with either homodimer, retains strong binding to activin A,GDF8, and GDF11 as observed with ActRIIB-Fc homodimer, and exhibitssubstantially reduced binding to BMP9, BMP10, and GDF3. Like ActRIIB-Fchomodimer, the heterodimer retains intermediate-level binding to BMP6.

FIG. 7 shows a multiple sequence alignment of ALK4 extracellular domainsderived from various vertebrate species (SEQ ID NOs: 59-65).

FIGS. 8A-8D show schematic examples of heteromeric protein complexescomprising an ALK4 polypeptide (e.g. a polypeptide that is at least 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100%identical to an extracellular domain of an ALK4 protein from humans orother species such as those described herein, e.g., SEQ ID Nos: 9, 10,19, 20, 42, 44, 47, and 48) and an ActRIIB polypeptide (e.g. apolypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 97%, 98%, 99% or 100% identical to an extracellular domain ofan ActRIIB protein from humans or other species such as those describedherein, e.g., SEQ ID Nos: 1, 2, 3, 4, 5, 6, 39, 41, 45, and 46).

In the illustrated embodiments, the ALK4 polypeptide (from left toright) is part of a fusion polypeptide that comprises a first member ofan interaction pair (“C₁”), and the ActRIIB polypeptide is part of afusion polypeptide that comprises a second member of an interaction pair(“C₂”). Suitable interaction pairs included, for example, heavy chainand/or light chain immunoglobulin interaction pairs, truncations, andvariants thereof such as those described herein [e.g., Spiess et al(2015) Molecular Immunology 67(2A): 95-106]. In each fusion polypeptide,a linker may be positioned between the ALK4 or ActRIIB polypeptide andthe corresponding member of the interaction pair. The first and secondmembers of the interaction pair may be unguided, meaning that themembers of the pair may associate with each other or self-associatewithout substantial preference, and they may have the same or differentamino acid sequences. See FIG. 8A. Alternatively, the interaction pairmay be a guided (asymmetric) pair, meaning that the members of the pairassociate preferentially with each other rather than self-associate. SeeFIG. 8B. Complexes of higher order can be envisioned. See FIGS. 8C and8D.

FIGS. 9A-9G show schematic examples of heteromeric protein complexescomprising two ALK4 polypeptides (e.g. polypeptide that areindependently at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,97%, 98%, 99% or 100% identical to an extracellular domain of an ALK4protein from humans or other species such as those described herein,e.g., SEQ ID Nos: 9, 10, 19, 20, 42, 44, 47, and 48) and two ActRIIBpolypeptides (e.g. two polypeptides that are independently at least 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100%identical to an extracellular domain of an ActRIIB protein from humansor other species such as those described herein, e.g., SEQ ID Nos: 1, 2,3, 4, 5, 6, 39, 41, 45, and 46).

In the illustrated embodiment FIG. 9A, the first ALK4 polypeptide (fromleft to right) is part of a fusion polypeptide that comprises a firstmember of an interaction pair (“C₁”) and further comprises an additionalfirst member of an interaction pair (“A₁”); and the second ALK4polypeptide is part of a fusion polypeptide that comprises a secondmember of an interaction pair (“C₂”) and further comprises an firstmember of an interaction pair (“A₂”). The first ActRIIB polypeptide(from left to right) is part of a fusion polypeptide that comprises asecond member of an interaction pair (“B₁”); and the second ActRIIBpolypeptide is part of a fusion polypeptide that comprises a secondmember of an interaction pair (“B₂”). A₁ and A₂ may be the same ordifferent; B₁ and B₂ may be the same or different, and C₁ and C₂ may bethe same or different. In each fusion polypeptide, a linker may bepositioned between the ALK4 or ActRIIB polypeptide and the correspondingmember of the interaction pair as well as between interaction pairs.FIG. 9A is an example of an association of unguided interaction pairs,meaning that the members of the pair may associate with each other orself-associate without substantial preference and may have the same ordifferent amino acid sequences.

In the illustrated embodiment FIG. 9B, the first ActRIIB polypeptide(from left to right) is part of a fusion polypeptide that comprises afirst member of an interaction pair (“C₁”) and further comprises anadditional first member of an interaction pair (“A₁”); and the secondActRIIB polypeptide is part of a fusion polypeptide that comprises asecond member of an interaction pair (“B₂”). The first ALK4 polypeptide(from left to right) is part of a fusion polypeptide that comprises asecond member of an interaction pair (“B₁”); and the second ALK4polypeptide is part of a fusion polypeptide that comprises a secondmember of an interaction pair (“C₂”) and further comprises a firstmember of an interaction pair (“A₂”). In each fusion polypeptide, alinker may be positioned between the ALK4 or ActRIIB polypeptide and thecorresponding member of the interaction pair as well as betweeninteraction pairs. FIG. 9B is an example of an association of guided(asymmetric) interaction pairs, meaning that the members of the pairassociate preferentially with each other rather than self-associate.

Suitable interaction pairs included, for example, heavy chain and/orlight chain immunoglobulin interaction pairs, truncations, and variantsthereof as described herein [e.g., Spiess et al (2015) MolecularImmunology 67(2A): 95-106]. Complexes of higher order can be envisioned.See FIGS. 9C-9F. Using similar methods (particularly those that employlight and/or heavy chain immunoglobulins, truncations, or variantsthereof), interaction pairs may be used to produce ALK4:ActRIIBheterodimers that resemble antibody Fab and F(ab′)₂ complexes [e.g.,Spiess et al (2015) Molecular Immunology 67(2A): 95-106]. See FIG. 9G.

FIGS. 10A and 10B show schematic examples of a heteromeric proteincomplex comprising an ALK4 polypeptide (e.g. a polypeptide that is atleast 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or100% identical to an extracellular domain of an ALK4 protein from humansor other species as described herein, e.g., SEQ ID Nos: 9, 10, 19, 20,42, 44, 47, and 48), an ActRIIB polypeptide (e.g. a polypeptide that isat least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%or 100% identical to an extracellular domain of an ActRIIB protein fromhumans or other species as such as those described herein, e.g., SEQ IDNos: 1, 2, 3, 4, 5, 6, 39, 41, 45, and 46), and a ligand-binding domainof an antibody (e.g., a ligand-binding domain derived from an antibodythat binds to one or more ALK4:ActRIIB-binding ligands). In theillustrated embodiments, the ALK4 polypeptide is part of a fusionpolypeptide that comprises a first member of an interaction pair (“C₁”),and further comprises an additional first member of an interaction pair(“A₁”). The ActRIIB polypeptide is part of a fusion polypeptide thatcomprises a second member of an interaction pair (“B₁”). The variableheavy chain (V_(H)) polypeptide is part of a fusion polypeptide thatcomprises a second member of an interaction pair (“C₂”), and furthercomprises a first member of an interaction pair (“A₂”). The variableheavy chain (V_(L)) polypeptide is part of a fusion polypeptide thatcomprises a second member of an interaction pair (“B₂”). In each fusionpolypeptide, a linker may be positioned between the ALK4 or ActRIIBpolypeptide and the corresponding member of the interaction pair,between interaction pairs, and between the V_(H) and V_(L) polypeptidesand a member of the interaction pair. A₁ and A₂ may be the same ordifferent; B₁ and B₂ may be the same or different, and C₁ and C₂ may bethe same or different. Suitable interaction pairs included, for example,constant heavy chain and/or light chain immunoglobulin interactionpairs, truncations, and variants thereof as described herein [e.g.,Spiess et al (2015) Molecular Immunology 67(2A): 95-106]. FIG. 10A is anexample of an association of guided (asymmetric) interaction pairs,meaning that the members of the pair associate preferentially with eachother rather than self-associate. FIG. 10B is an example of anassociation of unguided interaction pairs, meaning that the members ofthe pair may associate with each other or self-associate withoutsubstantial preference and may have the same or different amino acidsequences.

Such antibody-ALK4:ActRIIB complexes may be useful in situations whereit is desirable to further bind/antagonize an agent that is not anALK4:ActRIIB ligand. Alternatively, such antibody-ALK4:ActRIIB complexesmay be useful in situations where it is desirable to further enhanceALK4:ActRIIB ligand binding/antagonism. For example, as demonstrated bythe examples herein, activin B, activin A, GDF11, and GDF8 all bind withstrong affinity to an ALK4:ActRIIB heterodimer. In addition, BMP6 bindsto ALK4:ActRIIB heterodimers but with weaker affinity. In certainsituations where it is desirable to antagonize BMP6 activity, inaddition to one or more of the high affinity-binding ligands (e.g.,activin B, activin A, GDF11, and GDF8), BMP6 may be outcompeted forbinding to the ALK4:ActRIIB heterodimer. In such situations, addition ofBMP6-binding domain of an antibody to the ALK4:ActRIIB heteromultimercomplex would improve the capacity of such protein complexes toantagonize BMP6 in addition to one or more of activin B, activin A,GDF11, and GDF8.

FIG. 11 shows schematic examples of ALK4:ActRIIB single-trappolypeptides. ALK4:ActRIIB single-trap polypeptides may contain multipleALK4 domains (e.g., 1, 2, 3, 4, 5, 6, 7, 9, 10 or more domains), havingthe same or different sequences, and multiple ActRIIB domains (e.g., 1,2, 3, 4, 5, 6, 7, 9, 10 or more domains), having the same or differentsequences. These ALK4 and ActRIIB domains may be arranged in any orderand may comprise one or more linker domains positions between one ormore of the ALK4 and ActRIIB domains. Such ligand traps may be used astherapeutic agents to treat or prevent diseases or conditions describedherein.

FIG. 12A-12D show schematic examples of multimeric protein complexcomprising at least one ALK4:ActRIIB single-chain trap polypeptides. Inthe illustrated embodiments 12A and 12B, a first ALK4:ActRIIBsingle-chain trap polypeptide (from left to right) is part of a fusionpolypeptide that comprises a first member of an interaction pair (“C₁”);and a second ALK4:ActRIIB single-chain trap polypeptide is part of afusion polypeptide that comprises a second member of an interaction pair(“C₂”). C₁ and C₂ may be the same or different. The first and secondALK4:ActRIIB single-chain trap polypeptides may be the same ordifferent. In each fusion polypeptide, a linker may be positionedbetween the ALK4:ActRIIB single-chain trap polypeptide and thecorresponding member of the interaction pair. Suitable interaction pairsincluded, for example, heavy chain and/or light chain immunoglobulininteraction pairs, truncations, and variants thereof as described herein[e.g., Spiess et al (2015) Molecular Immunology 67(2A): 95-106]. FIG.12A is an example of an association of unguided interaction pairs,meaning that the members of the pair may associate with each other orself-associate without substantial preference and may have the same ordifferent amino acid sequences. FIG. 12B is an example of an associationof guided (asymmetric) interaction pairs, meaning that the members ofthe pair associate preferentially with each other rather thanself-associate. Complexes of higher order can be envisioned. Inaddition, such ALK4:ActRIIB single-chain trap polypeptides may besimilarly be associated, covalently or non-covalently, with one or moreALK4 polypeptides and/or one or more ActRIIB polypeptides. See FIG. 12C.Also, such ALK4:ActRIIB single-chain trap polypeptides may be similarlybe associated, covalently or non-covalently, with one or moreligand-binding domain of an antibody (e.g., a ligand-biding domain of anantibody that binds to one or more ALK4:ActRIIB binding ligands). SeeFIG. 12D.

FIG. 13 shows comparative ALK4-Fc:ActRIIB-Fcheterodimer/ActRIIB-Fc:ActRIIB-Fc homodimer IC₅₀ data as determined byan A-204 Reporter Gene Assay as described herein. ALK4-Fc:ActRIIB-Fcheterodimer inhibits activin A, activin B, GDF8, and GDF11 signalingpathways similarly to the ActRIIB-Fc:ActRIIB-Fc homodimer. However,ALK4-Fc:ActRIIB-Fc heterodimer inhibition of BMP9 and BMP10 signalingpathways is significantly reduced compared to the ActRIIB-Fc:ActRIIB-Fchomodimer. These data demonstrate that ALK4:ActRIIB heterodimers aremore selective antagonists of activin A, activin B, GDF8, and GDF11compared to corresponding ActRIIB:ActRIIB homodimers.

FIGS. 14A-14C shows gene expression profiles of fibrotic genes (Col1a1,Fibronectin, PAI-1, CTGF, and a-SMA), inflammatory genes (TNF-alpha, andMCP1), cytokine genes (TGF-beta 1, GF-beta 2, TGF-beta 3, and activinA), kidney injury gene (NGAL), Hypoxia-inducible factor 1-alpha (HIF1a),and activin A receptor (Acvr2A) from mouse kidneys subjected tounilateral ureteral obstruction (UUO). Samples from the contralateral,non-surgery kidney were used as a control (Ctrl). Gene expressionprofiles were obtained at 17 days post-surgery. Mice were administeredeither PBS or an ALK4-Fc:ActRIIB-Fc homodimer at days 3, 7, 10, and 14post-surgery. ($) denotes a statistical difference between UUO kidneysat 17 days in mice administered only PBS compared UUO kidneys at 17 daysin mice administered the ALK7-Fc:ActRIIB-Fc homodimer. (@) denotes thatno transcript was detected.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

In part, the present disclosure relates to heteromultimers comprising aTGFβ superfamily type I receptor polypeptide and a TGFβ superfamily typeII receptor polypeptide, uses thereof, and methods of making suchheteromultimers. See, e.g., FIGS. 1 and 2. In certain preferredembodiments, heteromultimers comprise an extracellular domain of a TGFβsuperfamily type I receptor polypeptide and an extracellular domain of aTGFβ superfamily type II receptor polypeptide. In particular, thedisclosure provides heteromultimers that comprise an ALK4 polypeptideand an ActRIIB polypeptide. Preferably such ALK4 polypeptides comprise aligand-binding domain of an ALK4 receptor and such ActRIIB polypeptidescomprise a ligand-binding domain of an ActRIIB receptor. In certainpreferred embodiments, ALK4:ActRIIB heteromultimers of the disclosurehave an altered TGFβ superfamily ligand binding profile/specificitycompared to a corresponding sample of a homomultimer (e.g., anALK4:ActRIIB heterodimer compared to an ActRIIB:ActRIIB homodimer or anALK4:ALK4 homodimer).

The TGF-β superfamily is comprised of over 30 secreted factors includingTGF-betas, activins, nodals, bone morphogenetic proteins (BMPs), growthand differentiation factors (GDFs), and anti-Mullerian hormone (AMH)[Weiss et al. (2013) Developmental Biology, 2(1): 47-63]. Members of thesuperfamily, which are found in both vertebrates and invertebrates, areubiquitously expressed in diverse tissues and function during theearliest stages of development throughout the lifetime of an animal.Indeed, TGF-β superfamily proteins are key mediators of stem cellself-renewal, gastrulation, differentiation, organ morphogenesis, andadult tissue homeostasis. Consistent with this ubiquitous activity,aberrant TGF-beta superfamily signaling is associated with a wide rangeof human pathologies including, for example, autoimmune disease,cardiovascular disease, fibrotic disease, and cancer.

Ligands of the TGF-beta superfamily share the same dimeric structure inwhich the central 3½ turn helix of one monomer packs against the concavesurface formed by the beta-strands of the other monomer. The majority ofTGF-beta family members are further stabilized by an intermoleculardisulfide bond. This disulfide bonds traverses through a ring formed bytwo other disulfide bonds generating what has been termed a ‘cysteineknot’ motif [Lin et al. (2006) Reproduction 132: 179-190; and Hinck etal. (2012) FEBS Letters 586: 1860-1870].

TGF-beta superfamily signaling is mediated by heteromeric complexes oftype I and type II serine/threonine kinase receptors, whichphosphorylate and activate downstream SMAD proteins (e.g., SMAD proteins1, 2, 3, 5, and 8) upon ligand stimulation [Massagué (2000) Nat. Rev.Mol. Cell Biol. 1:169-178]. These type I and type II receptors aretransmembrane proteins, composed of a ligand-binding extracellulardomain with cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine kinase specificity.In general, type I receptors mediate intracellular signaling while thetype II receptors are required for binding TGF-beta superfamily ligands.Type I and II receptors form a stable complex after ligand binding,resulting in phosphorylation of type I receptors by type II receptors.

The TGF-beta family can be divided into two phylogenetic branches basedon the type I receptors they bind and the Smad proteins they activate.One is the more recently evolved branch, which includes, e.g., theTGF-betas, activins, GDF8, GDF9, GDF11, BMP3 and nodal, which signalthrough type I receptors that activate Smads 2 and 3 [Hinck (2012) FEBSLetters 586:1860-1870]. The other branch comprises the more distantlyrelated proteins of the superfamily and includes, e.g., BMP2, BMP4,BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF1, GDF5, GDF6, and GDF7,which signal through Smads 1, 5, and 8.

TGF-beta isoforms are the founding members of the TGF-beta superfamily,of which there are 3 known isoforms in mammals designated as TGF-beta1,TGF-beta2 and TGF-beta3. Mature bioactive TGF-beta ligands function ashomodimers and predominantly signal through the type I receptor ALK5,but have also been found to additionally signal through ALK1 inendothelial cells [Goumans et al. (2003) Mol Cell 12(4): 817-828].TGF-beta1 is the most abundant and ubiquitously expressed isoform.TGF-beta1 is known to have an important role in wound healing, and miceexpressing a constitutively active TGF-beta1 transgene develop fibrosis[Clouthier et al. (1997) J Clin. Invest. 100(11): 2697-2713]. TGF-beta1is also involved in T cell activation and maintenance of T regulatorycells [Li et al. (2006) Immunity 25(3): 455-471]. TGF-beta2 expressionwas first described in human glioblastoma cells, and is occurs inneurons and astroglial cells of the embryonic nervous system. TGF-beta2is known to suppress interleukin-2-dependent growth of T lymphocytes.TGF-beta3 was initially isolated from a human rhabdomyosarcoma cell lineand since has been found in lung adenocarcinoma and kidney carcinomacell lines. TGF-beta3 is known to be important for palate and lungmorphogenesis [Kubiczkova et al. (2012) Journal of TranslationalMedicine 10:183].

Activins are members of the TGF-beta superfamily and were initiallydiscovered as regulators of secretion of follicle-stimulating hormone,but subsequently various reproductive and non-reproductive roles havebeen characterized. There are three principal activin forms (A, B, andAB) that are homo/heterodimers of two closely related β subunits(β_(A)β_(A), β_(B)β_(B), and β_(A)β_(B), respectively). The human genomealso encodes an activin C and an activin E, which are primarilyexpressed in the liver, and heterodimeric forms containing β_(C) orβ_(E) are also known. In the TGF-beta superfamily, activins are uniqueand multifunctional factors that can stimulate hormone production inovarian and placental cells, support neuronal cell survival, influencecell-cycle progress positively or negatively depending on cell type, andinduce mesodermal differentiation at least in amphibian embryos [DePaoloet al. (1991) Proc Soc Ep Biol Med. 198:500-512; Dyson et al. (1997)Curr Biol. 7:81-84; and Woodruff (1998) Biochem Pharmacol. 55:953-963].In several tissues, activin signaling is antagonized by its relatedheterodimer, inhibin. For example, in the regulation offollicle-stimulating hormone (FSH) secretion from the pituitary, activinpromotes FSH synthesis and secretion, while inhibin reduces FSHsynthesis and secretion. Other proteins that may regulate activinbioactivity and/or bind to activin include follistatin (FS),follistatin-related protein (FSRP, also known as FLRG or FSTL3), andα₂-macroglobulin.

As described herein, agents that bind to “activin A” are agents thatspecifically bind to the β_(A) subunit, whether in the context of anisolated β_(A) subunit or as a dimeric complex (e.g., a β_(A)β_(A)homodimer or a β_(A)β_(B) heterodimer). In the case of a heterodimercomplex (e.g., a β_(A)β_(B) heterodimer), agents that bind to “activinA” are specific for epitopes present within the β_(A) subunit, but donot bind to epitopes present within the non-β_(A) subunit of the complex(e.g., the β_(B) subunit of the complex). Similarly, agents disclosedherein that antagonize (inhibit) “activin A” are agents that inhibit oneor more activities as mediated by a β_(A) subunit, whether in thecontext of an isolated β_(A) subunit or as a dimeric complex (e.g., aβ_(A)β_(A) homodimer or a β_(A)β_(B) heterodimer). In the case ofβ_(A)β_(B) heterodimers, agents that inhibit “activin A” are agents thatspecifically inhibit one or more activities of the β_(A) subunit, but donot inhibit the activity of the non-β_(A) subunit of the complex (e.g.,the β_(B) subunit of the complex). This principle applies also to agentsthat bind to and/or inhibit “activin B”, “activin C”, and “activin E”.Agents disclosed herein that antagonize “activin AB” are agents thatinhibit one or more activities as mediated by the β_(A) subunit and oneor more activities as mediated by the β_(B) subunit. The same principlealso applies to agent that bind to and/or inhibit “activin AC”, “activinBC”, “activin AE”, and “activin BE”.

Nodal proteins have functions in mesoderm and endoderm induction andformation, as well as subsequent organization of axial structures suchas heart and stomach in early embryogenesis. It has been demonstratedthat dorsal tissue in a developing vertebrate embryo contributespredominantly to the axial structures of the notochord and pre-chordalplate while it recruits surrounding cells to form non-axial embryonicstructures. Nodal appears to signal through both type I and type IIreceptors and intracellular effectors known as SMAD proteins. Studiessupport the idea that ActRIIA and ActRIIB serve as type II receptors forNodal [Sakuma et al. (2002) Genes Cells. 2002, 7:401-12]. It issuggested that Nodal ligands interact with their co-factors (e.g.,Crypto or Cryptic) to activate activin type I and type II receptors,which phosphorylate SMAD2. Nodal proteins are implicated in many eventscritical to the early vertebrate embryo, including mesoderm formation,anterior patterning, and left-right axis specification. Experimentalevidence has demonstrated that nodal signaling activates pAR3-Lux, aluciferase reporter previously shown to respond specifically to activinand TGF-beta. However, nodal is unable to induce pT1×2-Lux, a reporterspecifically responsive to bone morphogenetic proteins. Recent resultsprovide direct biochemical evidence that Nodal signaling is mediated bySMAD2 and SMAD3, which also mediate signaling by TGF-betas and activins.Further evidence has shown that the extracellular protein Cripto orCryptic is required for nodal signaling, making it distinct from activinor TGF-beta signaling.

The BMPs and GDFs together form a family of cysteine-knot cytokinessharing the characteristic fold of the TGF-beta superfamily [Rider etal. (2010) Biochem J., 429(1):1-12]. This family includes, for example,BMP2, BMP4, BMP6, BMP7, BMP2a, BMP3, BMP3b (also known as GDF10), BMP4,BMP5, BMP6, BMP7, BMP8, BMP8a, BMP8b, BMP9 (also known as GDF2), BMP10,BMP11 (also known as GDF11), BMP12 (also known as GDF7), BMP13 (alsoknown as GDF6), BMP14 (also known as GDF5), BMP15, GDF1, GDF3 (alsoknown as VGR2), GDF8 (also known as myostatin), GDF9, GDF15, anddecapentaplegic. Besides the ability to induce bone formation, whichgave the BMPs their name, the BMP/GDFs display morphogenetic activitiesin the development of a wide range of tissues. BMP/GDF homo- andhetero-dimers interact with combinations of type I and type II receptordimers to produce multiple possible signaling complexes, leading to theactivation of one of two competing sets of SMAD transcription factors.BMP/GDFs have highly specific and localized functions. These areregulated in a number of ways, including the developmental restrictionof BMP/GDF expression and through the secretion of several specific BMPantagonist proteins that bind with high affinity to the cytokines.Curiously, a number of these antagonists resemble TGF-beta superfamilyligands.

Growth and differentiation factor-8 (GDF8) is also known as myostatin.GDF8 is a negative regulator of skeletal muscle mass and is highlyexpressed in developing and adult skeletal muscle. The GDF8 nullmutation in transgenic mice is characterized by a marked hypertrophy andhyperplasia of skeletal muscle [McPherron et al. Nature (1997)387:83-90]. Similar increases in skeletal muscle mass are evident innaturally occurring mutations of GDF8 in cattle and, strikingly, inhumans [Ashmore et al. (1974) Growth, 38:501-507; Swatland and Kieffer,J. Anim. Sci. (1994) 38:752-757; McPherron and Lee, Proc. Natl. Acad.Sci. USA (1997) 94:12457-12461; Kambadur et al. Genome Res. (1997)7:910-915; and Schuelke et al. (2004) N Engl J Med, 350:2682-8]. Studieshave also shown that muscle wasting associated with HIV-infection inhumans is accompanied by increases in GDF8 protein expression[Gonzalez-Cadavid et al., PNAS (1998) 95:14938-43]. In addition, GDF8can modulate the production of muscle-specific enzymes (e.g., creatinekinase) and modulate myoblast cell proliferation [International PatentApplication Publication No. WO 00/43781]. The GDF8 propeptide cannoncovalently bind to the mature GDF8 domain dimer, inactivating itsbiological activity [Miyazono et al. (1988) J. Biol. Chem., 263:6407-6415; Wakefield et al. (1988) J. Biol. Chem., 263; 7646-7654; andBrown et al. (1990) Growth Factors, 3: 35-43]. Other proteins which bindto GDF8 or structurally related proteins and inhibit their biologicalactivity include follistatin, and potentially, follistatin-relatedproteins [Gamer et al. (1999) Dev. Biol., 208: 222-232].

GDF11, also known as BMP11, is a secreted protein that is expressed inthe tail bud, limb bud, maxillary and mandibular arches, and dorsal rootganglia during mouse development [McPherron et al. (1999) Nat. Genet.,22: 260-264; and Nakashima et al. (1999) Mech. Dev., 80: 185-189]. GDF11plays a unique role in patterning both mesodermal and neural tissues[Gamer et al. (1999) Dev Biol., 208:222-32]. GDF11 was shown to be anegative regulator of chondrogenesis and myogenesis in developing chicklimb [Gamer et al. (2001) Dev Biol., 229:407-20]. The expression ofGDF11 in muscle also suggests its role in regulating muscle growth in asimilar way to GDF8. In addition, the expression of GDF11 in brainsuggests that GDF11 may also possess activities that relate to thefunction of the nervous system. Interestingly, GDF11 was found toinhibit neurogenesis in the olfactory epithelium [Wu et al. (2003)Neuron., 37:197-207]. Hence, inhibitors GDF11 may have in vitro and invivo applications in the treatment of diseases such as muscle diseasesand neurodegenerative diseases (e.g., amyotrophic lateral sclerosis).

BMP7, also called osteogenic protein-1 (OP-1), is well known to inducecartilage and bone formation. In addition, BMP7 regulates a wide arrayof physiological processes. For example, BMP7 may be the osteoinductivefactor responsible for the phenomenon of epithelial osteogenesis. It isalso found that BMP7 plays a role in calcium regulation and bonehomeostasis. Like activin, BMP7 binds to type II receptors, ActRIIA andActRIIB However, BMP7 and activin recruit distinct type I receptors intoheteromeric receptor complexes. The major BMP7 type I receptor observedwas ALK2, while activin bound exclusively to ALK4 (ActRIIB) BMP7 andactivin elicited distinct biological responses and activated differentSMAD pathways [Macias-Silva et al. (1998) J Biol Chem. 273:25628-36].

As described herein, comparative binding data demonstrated that anALK4:ActRIIB heterodimer has an altered binding profile (ligandselectivity) compared to either corresponding ActRIIB or ALK4homodimers. In particular, the ALK4:ActRIIB heterodimer displaysenhanced binding to activin B compared with either homodimer, andretains strong binding to activin A, GDF8, and GDF11 as observed withthe ActRIIB homodimer. However, the ALK4:ActRIIB heterodimer exhibitssubstantially reduced binding to BMP9, BMP10, and GDF3 compared to theActRIIB homodimer. In particular, BMP9 displays low or no observableaffinity for the ALK4:ActRIIB heterodimer, whereas this ligand bindsstrongly to ActRIIB homodimer.

These results therefore demonstrate that ALK4:ActRIIB heterodimers aremore selective antagonists of activin A, activin B, GDF8, and GDF11compared to ActRIIB homodimers. Accordingly, an ALK4:ActRIIB heterodimerwill be more useful than an ActRIIB homodimer in certain applicationswhere such selective antagonism is advantageous. Examples includetherapeutic applications where it is desirable to retain antagonism ofone or more of activin (e.g., activin A, activin B, activin AC, activinAB), GDF8, and GDF11 but minimize antagonism of one or more of BMP9,BMP10, and BMP6.

Moreover, ALK4:ActRIIB heterodimers, as described herein, exertbeneficial anabolic effects on skeletal muscle and bone as well ascatabolic effects on adipose tissue, very similar to those of an ActRIIBhomodimer. However, unlike ActRIIB homodimer, an ActRIIB:ALK4heterodimer exhibits only low-affinity or transient binding to BMP9 andBMP10 and so will have little to no concurrent inhibition on processesmediated by those ligands, such as angiogenesis. This novel selectivitywill be useful, for example, in treating patients in need of stimulatoryeffects on, e.g., muscle and bone, and inhibitory effects on fat, butnot in need of altered angiogenesis.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this disclosure and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification to provide additional guidanceto the practitioner in describing the compositions and methods of thedisclosure and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which it isused.

The terms “heteromer” or “heteromultimer” is a complex comprising atleast a first polypeptide chain and a second polypeptide chain, whereinthe second polypeptide chain differs in amino acid sequence from thefirst polypeptide chain by at least one amino acid residue. Theheteromer can comprise a “heterodimer” formed by the first and secondpolypeptide chains or can form higher order structures where one or morepolypeptide chains in addition to the first and second polypeptidechains are present. Exemplary structures for the heteromultimer includeheterodimers, heterotrimers, heterotetramers and further oligomericstructures. Heterodimers are designated herein as X:Y or equivalently asX-Y, where X represents a first polypeptide chain and Y represents asecond polypeptide chain. Higher-order heteromers and oligomericstructures are designated herein in a corresponding manner. In certainembodiments a heteromultimer is recombinant (e.g., one or morepolypeptide components may be a recombinant protein), isolated and/orpurified.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions. However, in common usage andin the instant application, the term “homologous,” when modified with anadverb such as “highly,” may refer to sequence similarity and may or maynot relate to a common evolutionary origin.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

“Percent (%) sequence identity” with respect to a reference polypeptide(or nucleotide) sequence is defined as the percentage of amino acidresidues (or nucleic acids) in a candidate sequence that are identicalto the amino acid residues (or nucleic acids) in the referencepolypeptide (nucleotide) sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid (nucleic acid) sequenceidentity values are generated using the sequence comparison computerprogram ALIGN-2. The ALIGN-2 sequence comparison computer program wasauthored by Genentech, Inc., and the source code has been filed withuser documentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available from Genentech, Inc., SouthSan Francisco, Calif., or may be compiled from the source code. TheALIGN-2 program should be compiled for use on a UNIX operating system,including digital UNIX V4.0D. All sequence comparison parameters are setby the ALIGN-2 program and do not vary.

As used herein, unless otherwise stated, “does not substantially bind toX” is intended to mean that an agent has a K_(D) that is greater thanabout 10⁻⁷, 10⁻⁶, 10⁻⁵, 10⁻⁴ or greater (e.g., no detectable binding bythe assay used to determine the K_(D)) for “X”, wherein “X” is aspecified agent such as a protein or nucleic acid.

“Agonize”, in all its grammatical forms, refers to the process ofactivating a protein and/or gene (e.g., by activating or amplifying thatprotein's gene expression or by inducing an inactive protein to enter anactive state) or increasing a protein's and/or gene's activity.

“Antagonize”, in all its grammatical forms, refers to the process ofinhibiting a protein and/or gene (e.g., by inhibiting or decreasing thatprotein's gene expression or by inducing an active protein to enter aninactive state) or decreasing a protein's and/or gene's activity.

The terms “about” and “approximately” as used in connection with anumerical value throughout the specification and the claims denotes aninterval of accuracy, familiar and acceptable to a person skilled in theart. In general, such interval of accuracy is ±10%. Alternatively, andparticularly in biological systems, the terms “about” and“approximately” may mean values that are within an order of magnitude,preferably ≤5-fold and more preferably ≤2-fold of a given value.

Numeric ranges disclosed herein are inclusive of the numbers definingthe ranges.

The terms “a” and “an” include plural referents unless the context inwhich the term is used clearly dictates otherwise. The terms “a” (or“an”), as well as the terms “one or more,” and “at least one” can beused interchangeably herein. Furthermore, “and/or” where used herein isto be taken as specific disclosure of each of the two or more specifiedfeatures or components with or without the other. Thus, the term“and/or” as used in a phrase such as “A and/or B” herein is intended toinclude “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, theterm “and/or” as used in a phrase such as “A, B, and/or C” is intendedto encompass each of the following aspects: A, B, and C; A, B, or C; Aor C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone);and C (alone).

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or groups of integers but not the exclusion of anyother integer or group of integers.

2. ALK4:ActRIIB Antagonists

As described herein, it has been discovered that an ALK4:ActRIIBheterodimers are unique antagonists of ligands of the TGF-betasuperfamily, exhibiting a different ligand-binding profile/selectivitycompared to corresponding ActRIIB and ALK4 homodimers. In particular, anexemplary ALK4:ActRIIB heterodimer displays enhanced binding to activinB compared to either homodimer, retains strong binding to activin A,GDF8, and GDF11 as observed with ActRIIB homodimer, but exhibitssubstantially reduced binding to BMP9, BMP10, and GDF3. In fact, theALK4:ActRIIB heterodimer displays low to no observable affinity forBMP9, whereas this ligand binds strongly to ActRIIB homodimer. See FIG.6. These results therefore demonstrate that ALK4:ActRIIB heterodimersare a more selective antagonists (inhibitors) of certain ligands of theTGF-beta superfamily compared to ActRIIB homodimers. Accordingly, anALK4:ActRIIB heterodimer will be more useful than an ActRIIB homodimerin certain applications where such selective antagonism is advantageous.Examples include therapeutic applications where it is desirable toantagonize one or more of activin (e.g., activin A, activin B, activinAB, activin AC), GDF8, and GDF11 with decreased antagonism of one ormore of BMP9, BMP10, and GDF3.

Moreover, ALK4:ActRIIB heterodimer produced certain biological effectsstrikingly similar to those of an ActRIIB homodimer despite differentialligand selectivity of the two complexes. For example, ALK4:ActRIIBheterodimer exerts beneficial anabolic effects on skeletal muscle andbone as well as catabolic effects on adipose tissue, very similar tothose of an ActRIIB-Fc homodimer. However, unlike ActRIIB homodimer,ActRIIB:ALK4 heterodimer exhibits only low-affinity or transient bindingto BMP9 and BMP10 and so should have little to no concurrent inhibitionon processes mediated by those ligands, such as angiogenesis. This novelselectivity may be useful, for example, in treating patients in need ofstimulatory effects on muscle and bone, and/or inhibitory effects onfat, but not in need of altered angiogenesis. Therefore, while notwishing to be bound to a particular mechanisms of action, it is expectedthat ALK4:ActRIIB heteromultimers, as well as variants thereof, thatbind to/inhibit at least one or more of ALK4:ActRIIB-binding ligandswill be useful agents for promoting beneficial anabolic effects onskeletal muscle and bone and catabolic effects on adipose tissue.Furthermore, it is expected that other antagonists (inhibitors), orcombinations of antagonists, that mimic the binding/inhibitoryproperties of the ALK4:ActRIIB heterodimers described herein as well asagents that directly or indirectly antagonize ALK4 and/or ActRIIBreceptors, agents that directly or indirectly antagonize ALK4- and/orActRIIB-binding ligands, agents that directly or indirectly antagonizedownstream signaling mediators (e.g., Smads), and/or agents thatdirectly or indirectly antagonize TGF-beta superfamily co-receptors willhave similar biological effects including, for example, stimulatoryeffects on muscle and bone and inhibitory effects on fat. Theseantagonistic mimetic are collectively referred to herein as“ALK4:ActRIIB antagonists” or “ALK4:ActRIIB inhibitors”.

A. ALK4:ActRIIB Heteromultimers

In certain aspects, the present disclosure relates to heteromultimerscomprising one or more ALK4 receptor polypeptides (e.g., SEQ ID NOs: 9,10, 19, 20, 42, 44, 47 and 48) and one or more ActRIIB receptorpolypeptides (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 39, 41, 45, and 46),which are generally referred to herein as “ALK4:ActRIIB heteromultimercomplexes” or “ALK4:ActRIIB heteromultimers”. Preferably, ALK4:ActRIIBheteromultimers of the disclosure are soluble, for example, aheteromultimer may comprises a soluble portion (domain) of an ALK4receptor and a soluble portion (domain) of an ActRIIB receptor. Ingeneral, the extracellular domains of ALK4 and ActRIIB correspond to asoluble portion of these receptors. Therefore, in some embodiments,heteromultimers of the disclosure comprise an extracellular domain of anALK4 receptor and an extracellular domain of an ActRIIB receptor.Example extracellular domains ALK4 and ActRIIB receptors are disclosedherein and such sequences, as well as fragments, functional variants,and modified forms thereof, may be used in accordance with theinventions of the disclosure (e.g., ALK4:ActRIIB heteromultimercompositions and uses thereof). ALK4:ActRIIB heteromultimers of thedisclosure include, e.g., heterodimers, heterotrimers, heterotetramersand higher order oligomeric structures. See, e.g., FIGS. 1, 2, and 8-10.In certain preferred embodiments, heteromultimers of the disclosure areALK4:ActRIIB heterodimers.

Preferably, ALK4:ActRIIB heteromultimers of the disclosure bind to oneor more TGF-beta superfamily ligands. In some embodiments, ALK4:ActRIIBheteromultimers may bind to one or more of activin (e.g., activin A,activin B, activin C, activin E, activin AC, activin AB, activin BC,activin AE, and activin BE), GDF8, GDF11, BMP6, GDF3, and BMP10. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin A. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin B. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin C. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin E. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin AB. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin AC. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin AE. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin BC. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin BE. In someembodiments, ALK4:ActRIIB heteromultimers bind to GDF11. In someembodiments, ALK4:ActRIIB heteromultimers bind to GDF8. In someembodiments, ALK4:ActRIIB heteromultimers bind to BMP6. In someembodiments, ALK4:ActRIIB heteromultimers bind to GDF3. In someembodiments, ALK4:ActRIIB heteromultimers bind to BMP10. In someembodiments, ALK4:ActRIIB heteromultimers do not bind to, or no notsubstantially bind to BMP9 (e.g., have indeterminate K_(a) or K_(d) dueto the transient nature of the interaction between BMP9 and anALK4:ActRIIB heteromultimer). In some embodiments, ALK4:ActRIIBheteromultimers binds with stronger affinity to activin B compared to acorresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIBheteromultimers binds with weaker affinity to GDF3 compared to acorresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIBheteromultimers binds with weaker affinity to BMP9 compared to acorresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIBheteromultimers binds with weaker affinity to BMP10 compared to acorresponding ActRIIB homomultimer. Optionally, ALK4:ActRIIBheteromultimers may further bind to one or more of BMP2, BMP2/7, BMP3,BMP4, BMP4/7, BMP5, BMP7, BMP8a, BMP8b, GDF5, GDF6/BMP13, GDF7,GDF9b/BMP15, GDF15/MIC1, TGF-β1, TGF-β2, TGF-β3, nodal, glialcell-derived neurotrophic factor (GDNF), neurturin, artemin, persephin,MIS, and Lefty.

In some embodiments, ALK4:ActRIIB heteromultimers may be used to inhibit(antagonize) signaling (e.g., Smad 2/3 and/or Smad 1/5/8 signaling)mediated by one or more TGFβ superfamily ligands. In particular,ALK4:ActRIIB heteromultimers of the disclosure may be used to inhibitintracellular signaling by one or more TGFβ superfamily ligands in, forexample, a cell-based assay such as those described herein. For example,ALK4:ActRIIB heteromultimers may inhibit signaling mediated by one ormore of activin (e.g., activin A, activin B, activin C, activin E,activin AC, activin AB, activin BC, activin AE, and activin BE), GDF8,GDF11, BMP6, GDF3, and BMP10 in a cell-based assay. In some embodiments,ALK4:ActRIIB heteromultimers may inhibit activin A signaling in acell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers mayinhibit activin B signaling in a cell-based assay. In some embodiments,ALK4:ActRIIB heteromultimers may inhibit activin C signaling in acell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers mayinhibit activin D signaling in a cell-based assay. In some embodiments,ALK4:ActRIIB heteromultimers may inhibit activin E signaling in acell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers mayinhibit activin AB signaling in a cell-based assay. In some embodiments,ALK4:ActRIIB heteromultimers may inhibit activin AC signaling in acell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers mayinhibit activin BC signaling in a cell-based assay. In some embodiments,ALK4:ActRIIB heteromultimers may inhibit activin AE signaling in acell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers mayinhibit activin BE signaling in a cell-based assay. In some embodiments,ALK4:ActRIIB heteromultimers may inhibit GDF11 signaling in a cell-basedassay. In some embodiments, ALK4:ActRIIB heteromultimers may inhibitGDF8 signaling in a cell-based assay. In some embodiments, ALK4:ActRIIBheteromultimers may inhibit BMP6 signaling in a cell-based assay. Insome embodiments, ALK4:ActRIIB heteromultimers may inhibit GDF3signaling in a cell-based assay. In some embodiments, ALK4:ActRIIBheteromultimers may inhibit BMP9 signaling in a cell-based assay. Insome embodiments, ALK4:ActRIIB heteromultimers do not inhibit, or do notsubstantially inhibit BMP9 signaling in a cell-based assay. In someembodiments, ALK4:ActRIIB heteromultimers are stronger inhibitors ofactivin B signaling in a cell-based assay compared to a correspondingActRIIB homomultimer. In some embodiments, ALK4:ActRIIB heteromultimersare weaker inhibitors of BMP10 signaling in a cell-based assay comparedto a corresponding ActRIIB homomultimer. In some embodiments,ALK4:ActRIIB heteromultimers are stronger inhibitors of GDF3 signalingin a cell-based assay compared to a corresponding ActRIIB homomultimer.In some embodiments, ALK4:ActRIIB heteromultimers are strongerinhibitors of BMP9 signaling in a cell-based assay compared to acorresponding ActRIIB homomultimer. Optionally, ALK4:ActRIIBheteromultimers may further inhibit intracellular signaling by one ormore of BMP2, BMP2/7, BMP3, BMP4, BMP4/7, BMP5, BMP7, BMP8a, BMP8b,GDF5, GDF6/BMP13, GDF7, GDF9b/BMP15, GDF15/MIC1, TGF-β1, TGF-β2, TGF-β3,nodal, glial cell-derived neurotrophic factor (GDNF), neurturin,artemin, persephin, MIS, and Lefty in a cell-based assay.

As used herein, the term “ActRIIB” refers to a family of activinreceptor type IIB (ActRIIB) proteins from any species and variantsderived from such ActRIIB proteins by mutagenesis or other modification.Reference to ActRIIB herein is understood to be a reference to any oneof the currently identified forms. Members of the ActRIIB family aregenerally transmembrane proteins, composed of a ligand-bindingextracellular domain comprising a cysteine-rich region, a transmembranedomain, and a cytoplasmic domain with predicted serine/threonine kinaseactivity.

The term “ActRIIB polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ActRIIB family member as well asany variants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Examples of suchvariant ActRIIB polypeptides are provided throughout the presentdisclosure as well as in International Patent Application PublicationNos. WO 2006/012627, WO 2008/097541, and WO 2010/151426, which areincorporated herein by reference in their entirety. Numbering of aminoacids for all ActRIIB-related polypeptides described herein is based onthe numbering of the human ActRIIB precursor protein sequence providedbelow (SEQ ID NO: 1), unless specifically designated otherwise.

The human ActRIIB precursor protein sequence is as follows:

(SEQ ID NO: 1)   1 MTAPWVALAL LWGSLCAGS G RGEAETRECI YYNANWELER T NQSGLERCE  51 GEQDKRLHCY ASWR N SSGTI ELVKKGCWLD DFNCYDRQEC VATEENPQVY101 FCCCEGNFCN ERFTHLPEAG GPEVTYEPPP TAPTLLTVLA YSLLPIGGLS151 LIVLLAFWMY RHRKPPYGHV DIHEDPGPPP PSPLVGLKPL QLLEIKARGR201 FGCVWKAQLM NDFVAVKIFP LQDKQSWQSE REIFSTPGMK HENLLQFIAA251 EKRGSNLEVE LWLITAFHDK GSLTDYLKGN IITWNELCHV AETMSRGLSY301 LHEDVPWCRG EGHKPSIAHR DFKSKNVLLK SDLTAVLADF GLAVRFEPGK351 PPGDTHGQVG TRRYMAPEVL EGAINFQRDA FLRIDMYAMG LVLWELVSRC401 KAADGPVDEY MLPFEEEIGQ HPSLEELQEV VVHKKMRPTI KDHWLKHPGL451 AQLCVTIEEC WDHDAEARLS AGCVEERVSL IRRSVNGTTS DCLVSLVTSV501 TNVDLPPKES SI

The signal peptide is indicated with a single underline; theextracellular domain is indicated in bold font; and the potential,endogenous N-linked glycosylation sites are indicated with a doubleunderline.

The processed (mature) extracellular ActRIIB polypeptide sequence is asfollows:

(SEQ ID NO: 2) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTH LPEAGGPEVTYEPPPTAPT

In some embodiments, the protein may be produced with an “SGR . . . ”sequence at the N-terminus. The C-terminal “tail” of the extracellulardomain is indicated by a single underline. The sequence with the “tail”deleted (a Δ15 sequence) is as follows:

(SEQ ID NO: 3) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTH LPEA

A form of ActRIIB with an alanine at position 64 of SEQ ID NO: 1 (A64)is also reported in the literature See, e.g., Hilden et al. (1994)Blood, 83(8): 2163-2170. Applicants have ascertained that an ActRIIB-Fcfusion protein comprising an extracellular domain of ActRIIB with theA64 substitution has a relatively low affinity for activin and GDF11. Bycontrast, the same ActRIIB-Fc fusion protein with an arginine atposition 64 (R64) has an affinity for activin and GDF11 in the lownanomolar to high picomolar range. Therefore, sequences with an R64 areused as the “wild-type” reference sequence for human ActRIIB in thisdisclosure.

The form of ActRIIB with an alanine at position 64 is as follows:

(SEQ ID NO: 4)   1 MTAPWVALAL LWGSLCAGSG RGEAETRECI YYNANWELER TNQSGLERCE 51 GEQDKRLHCY ASWANSSGTI ELVKKGCWLD DFNCYDRQEC VATEENPQVY101 FCCCEGNFCN ERFTHLPEAG GPEVTYEPPP TAPTLLTVLA YSLLPIGGLS151 LIVLLAFWMY RHRKPPYGHV DIHEDPGPPP PSPLVGLKPL QLLEIKARGR201 FGCVWKAQLM NDFVAVKIFP LQDKQSWQSE REIFSTPGMK HENLLQFIAA251 EKRGSNLEVE LWLITAFHDK GSLTDYLKGN IITWNELCHV AETMSRGLSY301 LHEDVPWCRG EGHKPSIAHR DFKSKNVLLK SDLTAVLADF GLAVRFEPGK351 PPGDTHGQVG TRRYMAPEVL EGAINFQRDA FLRIDMYAMG LVLWELVSRC401 KAADGPVDEY MLPFEEEIGQ HPSLEELQEV VVHKKMRPTI KDHWLKHPGL451 AQLCVTIEEC WDHDAEARLS AGCVEERVSL IRRSVNGTTS DCLVSLVTSV501 TNVDLPPKES SI

The signal peptide is indicated by single underline and theextracellular domain is indicated by bold font.

The processed (mature) extracellular ActRIIB polypeptide sequence of thealternative A64 form is as follows:

(SEQ ID NO: 5) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTH LPEAGGPEVTYEPPPTAPT

In some embodiments, the protein may be produced with an “SGR . . . ”sequence at the N-terminus. The C-terminal “tail” of the extracellulardomain is indicated by single underline. The sequence with the “tail”deleted (a Δ15 sequence) is as follows:

(SEQ ID NO: 6) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTH LPEA

A nucleic acid sequence encoding the human ActRIIB precursor protein isshown below (SEQ ID NO: 7), representing nucleotides 25-1560 of GenbankReference Sequence NM_001106.3, which encode amino acids 1-513 of theActRIIB precursor. The sequence as shown provides an arginine atposition 64 and may be modified to provide an alanine instead. Thesignal sequence is underlined.

(SEQ ID NO: 7)   1 ATGACGGCGC CCTGGGTGGC CCTCGCCCTC CTCTGGGGAT CGCTGTGCGC  51 CGGCTCTGGG CGTGGGGAGG CTGAGACACG GGAGTGCATC TACTACAACG 101 CCAACTGGGA GCTGGAGCGC ACCAACCAGA GCGGCCTGGA GCGCTGCGAA 151 GGCGAGCAGG ACAAGCGGCT GCACTGCTAC GCCTCCTGGC GCAACAGCTC 201 TGGCACCATC GAGCTCGTGA AGAAGGGCTG CTGGCTAGAT GACTTCAACT 251 GCTACGATAG GCAGGAGTGT GTGGCCACTG AGGAGAACCC CCAGGTGTAC 301 TTCTGCTGCT GTGAAGGCAA CTTCTGCAAC GAACGCTTCA CTCATTTGCC 351 AGAGGCTGGG GGCCCGGAAG TCACGTACGA GCCACCCCCG ACAGCCCCCA 401 CCCTGCTCAC GGTGCTGGCC TACTCACTGC TGCCCATCGG GGGCCTTTCC 451 CTCATCGTCC TGCTGGCCTT TTGGATGTAC CGGCATCGCA AGCCCCCCTA 501 CGGTCATGTG GACATCCATG AGGACCCTGG GCCTCCACCA CCATCCCCTC 551 TGGTGGGCCT GAAGCCACTG CAGCTGCTGG AGATCAAGGC TCGGGGGCGC 601 TTTGGCTGTG TCTGGAAGGC CCAGCTCATG AATGACTTTG TAGCTGTCAA 651 GATCTTCCCA CTCCAGGACA AGCAGTCGTG GCAGAGTGAA CGGGAGATCT 701 TCAGCACACC TGGCATGAAG CACGAGAACC TGCTACAGTT CATTGCTGCC 751 GAGAAGCGAG GCTCCAACCT CGAAGTAGAG CTGTGGCTCA TCACGGCCTT 801 CCATGACAAG GGCTCCCTCA CGGATTACCT CAAGGGGAAC ATCATCACAT 851 GGAACGAACT GTGTCATGTA GCAGAGACGA TGTCACGAGG CCTCTCATAC 901 CTGCATGAGG ATGTGCCCTG GTGCCGTGGC GAGGGCCACA AGCCGTCTAT 951 TGCCCACAGG GACTTTAAAA GTAAGAATGT ATTGCTGAAG AGCGACCTCA1001 CAGCCGTGCT GGCTGACTTT GGCTTGGCTG TTCGATTTGA GCCAGGGAAA1051 CCTCCAGGGG ACACCCACGG ACAGGTAGGC ACGAGACGGT ACATGGCTCC1101 TGAGGTGCTC GAGGGAGCCA TCAACTTCCA GAGAGATGCC TTCCTGCGCA1151 TTGACATGTA TGCCATGGGG TTGGTGCTGT GGGAGCTTGT GTCTCGCTGC1201 AAGGCTGCAG ACGGACCCGT GGATGAGTAC ATGCTGCCCT TTGAGGAAGA1251 GATTGGCCAG CACCCTTCGT TGGAGGAGCT GCAGGAGGTG GTGGTGCACA1301 AGAAGATGAG GCCCACCATT AAAGATCACT GGTTGAAACA CCCGGGCCTG1351 GCCCAGCTTT GTGTGACCAT CGAGGAGTGC TGGGACCATG ATGCAGAGGC1401 TCGCTTGTCC GCGGGCTGTG TGGAGGAGCG GGTGTCCCTG ATTCGGAGGT1451 CGGTCAACGG CACTACCTCG GACTGTCTCG TTTCCCTGGT GACCTCTGTC1501 ACCAATGTGG ACCTGCCCCC TAAAGAGTCA AGCATC

A nucleic acid sequence encoding processed extracellular human ActRIIBpolypeptide is as follows (SEQ ID NO: 8). The sequence as shown providesan arginine at position 64, and may be modified to provide an alanineinstead.

(SEQ ID NO: 8)  1 GGGCGTGGGG AGGCTGAGAC ACGGGAGTGC ATCTACTACA ACGCCAACTG 51 GGAGCTGGAG CGAGCCACCC AGAGCGGCCT GGAGCGCTGC GAAGGCGAGC101 AGGACAAGCG GCTGCACTGC TACGCCTCCT GGCGCAACAG CTCTGGCACC151 ATCGAGCTCG TGAAGAAGGG CTGCTGGCTA GATGACTTCA ACTGCTACGA201 TAGGCAGGAG TGTGTGGCCA CTGAGGAGAA CCCCCAGGTG TACTTCTGCT251 GCTGTGAAGG CAACTTCTGC AACGAACGCT TCACTCATTT GCCAGAGGCT301 GGGGGCCCGG AAGTCACGTA CGAGCCACCC CCGACAGCCC CCACC

An alignment of the amino acid sequences of human ActRIIB extracellulardomain and human ActRIIA extracellular domain are illustrated in FIG. 3.This alignment indicates amino acid residues within both receptors thatare believed to directly contact ActRII ligands. For example, thecomposite ActRII structures indicated that the ActRIIB-ligand bindingpocket is defined, in part, by residues Y31, N33, N35, L38 through T41,E47, E50, Q53 through K55, L57, H58, Y60, S62, K74, W78 through N83,Y85, R87, A92, and E94 through F101. At these positions, it is expectedthat conservative mutations will be tolerated.

In addition, ActRIIB is well-conserved among vertebrates, with largestretches of the extracellular domain completely conserved. For example,FIG. 4 depicts a multi-sequence alignment of a human ActRIIBextracellular domain compared to various ActRIIB orthologs. Many of theligands that bind to ActRIIB are also highly conserved. Accordingly,from these alignments, it is possible to predict key amino acidpositions within the ligand-binding domain that are important for normalActRIIB-ligand binding activities as well as to predict amino acidpositions that are likely to be tolerant of substitution withoutsignificantly altering normal ActRIIB-ligand binding activities.Therefore, an active, human ActRIIB variant polypeptide useful inaccordance with the presently disclosed methods may include one or moreamino acids at corresponding positions from the sequence of anothervertebrate ActRIIB, or may include a residue that is similar to that inthe human or other vertebrate sequences. Without meaning to be limiting,the following examples illustrate this approach to defining an activeActRIIB variant. L46 in the human extracellular domain (SEQ ID NO: 53)is a valine in Xenopus ActRIIB (SEQ ID NO: 55), and so this position maybe altered, and optionally may be altered to another hydrophobicresidue, such as V, I or F, or a non-polar residue such as A. E52 in thehuman extracellular domain is a K in Xenopus, indicating that this sitemay be tolerant of a wide variety of changes, including polar residues,such as E, D, K, R, H, S, T, P, G, Y and probably A. T93 in the humanextracellular domain is a K in Xenopus, indicating that a widestructural variation is tolerated at this position, with polar residuesfavored, such as S, K, R, E, D, H, G, P, G and Y. F108 in the humanextracellular domain is a Y in Xenopus, and therefore Y or otherhydrophobic group, such as I, V or L should be tolerated. E111 in thehuman extracellular domain is K in Xenopus, indicating that chargedresidues will be tolerated at this position, including D, R, K and H, aswell as Q and N. R112 in the human extracellular domain is K in Xenopus,indicating that basic residues are tolerated at this position, includingR and H. A at position 119 in the human extracellular domain isrelatively poorly conserved, and appears as P in rodents and V inXenopus, thus essentially any amino acid should be tolerated at thisposition.

Moreover, ActRII proteins have been characterized in the art in terms ofstructural and functional characteristics, particularly with respect toligand binding [Attisano et al. (1992) Cell 68(1):97-108; Greenwald etal. (1999) Nature Structural Biology 6(1): 18-22; Allendorph et al.(2006) PNAS 103(20: 7643-7648; Thompson et al. (2003) The EMBO Journal22(7): 1555-1566; as well as U.S. Pat. Nos. 7,709,605, 7,612,041, and7,842,663]. In addition to the teachings herein, these referencesprovide amply guidance for how to generate ActRIIB variants that retainone or more normal activities (e.g., ligand-binding activity).

For example, a defining structural motif known as a three-finger toxinfold is important for ligand binding by type I and type II receptors andis formed by conserved cysteine residues located at varying positionswithin the extracellular domain of each monomeric receptor [Greenwald etal. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett586:1860-1870]. Accordingly, the core ligand-binding domains of humanActRIIB, as demarcated by the outermost of these conserved cysteines,corresponds to positions 29-109 of SEQ ID NO: 1 (ActRIIB precursor).Thus, the structurally less-ordered amino acids flanking thesecysteine-demarcated core sequences can be truncated by about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, or 28 residues at the N-terminus and/or by about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 residues at the C-terminus without necessarily alteringligand binding. Exemplary ActRIIB extracellular domains for N-terminaland/or C-terminal truncation include SEQ ID NOs: 2, 3, 5, and 6.

Attisano et al. showed that a deletion of the proline knot at theC-terminus of the extracellular domain of ActRIIB reduced the affinityof the receptor for activin. An ActRIIB-Fc fusion protein containingamino acids 20-119 of present SEQ ID NO: 1, “ActRIIB(20-119)-Fc”, hasreduced binding to GDF11 and activin relative to an ActRIIB(20-134)-Fc,which includes the proline knot region and the complete juxtamembranedomain (see, e.g., U.S. Pat. No. 7,842,663). However, anActRIIB(20-129)-Fc protein retains similar, but somewhat reducedactivity, relative to the wild-type, even though the proline knot regionis disrupted.

Thus, ActRIIB extracellular domains that stop at amino acid 134, 133,132, 131, 130 and 129 (with respect to SEQ ID NO: 1) are all expected tobe active, but constructs stopping at 134 or 133 may be most active.Similarly, mutations at any of residues 129-134 (with respect to SEQ IDNO: 1) are not expected to alter ligand-binding affinity by largemargins. In support of this, it is known in the art that mutations ofP129 and P130 (with respect to SEQ ID NO: 1) do not substantiallydecrease ligand binding. Therefore, an ActRIIB polypeptide of thepresent disclosure may end as early as amino acid 109 (the finalcysteine), however, forms ending at or between 109 and 119 (e.g., 109,110, 111, 112, 113, 114, 115, 116, 117, 118, or 119) are expected tohave reduced ligand binding. Amino acid 119 (with respect to present SEQID NO:1) is poorly conserved and so is readily altered or truncated.ActRIIB polypeptides ending at 128 (with respect to SEQ ID NO: 1) orlater should retain ligand-binding activity. ActRIIB polypeptides endingat or between 119 and 127 (e.g., 119, 120, 121, 122, 123, 124, 125, 126,or 127), with respect to SEQ ID NO: 1, will have an intermediate bindingability. Any of these forms may be desirable to use, depending on theclinical or experimental setting.

At the N-terminus of ActRIIB, it is expected that a protein beginning atamino acid 29 or before (with respect to SEQ ID NO: 1) will retainligand-binding activity. Amino acid 29 represents the initial cysteine.An alanine-to-asparagine mutation at position 24 (with respect to SEQ IDNO: 1) introduces an N-linked glycosylation sequence withoutsubstantially affecting ligand binding [U.S. Pat. No. 7,842,663]. Thisconfirms that mutations in the region between the signal cleavagepeptide and the cysteine cross-linked region, corresponding to aminoacids 20-29, are well tolerated. In particular, ActRIIB polypeptidesbeginning at position 20, 21, 22, 23, and 24 (with respect to SEQ IDNO: 1) should retain general ligand-biding activity, and ActRIIBpolypeptides beginning at positions 25, 26, 27, 28, and 29 (with respectto SEQ ID NO: 1) are also expected to retain ligand-biding activity. Ithas been demonstrated, e.g., U.S. Pat. No. 7,842,663, that,surprisingly, an ActRIIB construct beginning at 22, 23, 24, or 25 willhave the most activity.

Taken together, a general formula for an active portion (e.g.,ligand-binding portion) of ActRIIB comprises amino acids 29-109 of SEQID NO: 1. Therefore ActRIIB polypeptides may, for example, comprise,consist essentially of, or consist of an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of ActRIIBbeginning at a residue corresponding to any one of amino acids 20-29(e.g., beginning at any one of amino acids 20, 21, 22, 23, 24, 25, 26,27, 28, or 29) of SEQ ID NO: 1 and ending at a position corresponding toany one amino acids 109-134 (e.g., ending at any one of amino acids 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) of SEQ IDNO: 1. Other examples include polypeptides that begin at a position from20-29 (e.g., any one of positions 20, 21, 22, 23, 24, 25, 26, 27, 28, or29) or 21-29 (e.g., any one of positions 21, 22, 23, 24, 25, 26, 27, 28,or 29) of SEQ ID NO: 1 and end at a position from 119-134 (e.g., any oneof positions 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, or 134), 119-133 (e.g., any one of positions 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133),129-134 (e.g., any one of positions 129, 130, 131, 132, 133, or 134), or129-133 (e.g., any one of positions 129, 130, 131, 132, or 133) of SEQID NO: 1. Other examples include constructs that begin at a positionfrom 20-24 (e.g., any one of positions 20, 21, 22, 23, or 24), 21-24(e.g., any one of positions 21, 22, 23, or 24), or 22-25 (e.g., any oneof positions 22, 22, 23, or 25) of SEQ ID NO: 1 and end at a positionfrom 109-134 (e.g., any one of positions 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, or 134), 119-134 (e.g., any one of positions119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, or 134) or 129-134 (e.g., any one of positions 129, 130, 131, 132,133, or 134) of SEQ ID NO: 1. Variants within these ranges are alsocontemplated, particularly those having at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identity to the corresponding portion of SEQ ID NO: 1.

The variations described herein may be combined in various ways. In someembodiments, ActRIIB variants comprise no more than 1, 2, 5, 6, 7, 8, 9,10 or 15 conservative amino acid changes in the ligand-binding pocket,and zero, one, or more non-conservative alterations at positions 40, 53,55, 74, 79 and/or 82 in the ligand-binding pocket. Sites outside thebinding pocket, at which variability may be particularly well tolerated,include the amino and carboxy termini of the extracellular domain (asnoted above), and positions 42-46 and 65-73 (with respect to SEQ ID NO:1). An asparagine-to-alanine alteration at position 65 (N65A) actuallyimproves ligand binding in the A64 background, and is thus expected tohave no detrimental effect on ligand binding in the R64 background [U.S.Pat. No. 7,842,663]. This change probably eliminates glycosylation atN65 in the A64 background, thus demonstrating that a significant changein this region is likely to be tolerated. While an R64A change is poorlytolerated, R64K is well-tolerated, and thus another basic residue, suchas H may be tolerated at position 64 [U.S. Pat. No. 7,842,663].Additionally, the results of the mutagenesis program described in theart indicate that there are amino acid positions in ActRIIB that areoften beneficial to conserve. With respect to SEQ ID NO: 1, theseinclude position 80 (acidic or hydrophobic amino acid), position 78(hydrophobic, and particularly tryptophan), position 37 (acidic, andparticularly aspartic or glutamic acid), position 56 (basic amino acid),position 60 (hydrophobic amino acid, particularly phenylalanine ortyrosine). Thus, the disclosure provides a framework of amino acids thatmay be conserved in ActRIIB polypeptides. Other positions that may bedesirable to conserve are as follows: position 52 (acidic amino acid),position 55 (basic amino acid), position 81 (acidic), 98 (polar orcharged, particularly E, D, R or K), all with respect to SEQ ID NO: 1.

In certain embodiments, the disclosure relates to heteromultimers thatcomprise at least one ActRIIB polypeptide, which includes fragments,functional variants, and modified forms thereof. Preferably, ActRIIBpolypeptides for use in accordance with inventions of the disclosure aresoluble (e.g., an extracellular domain of ActRIIB) In other preferredembodiments, ActRIIB polypeptides for use in accordance with thedisclosure bind to one or more TGF-beta superfamily ligands. Therefore,in some embodiments, ActRIIB polypeptides for use in accordance with thedisclosure inhibit (antagonize) activity (e.g., inhibition of Smadsignaling) of one or more TGF-beta superfamily ligands. In someembodiments, heteromultimers of the disclosure comprise at least oneActRIIB polypeptide that comprise, consist essentially of, or consist ofan amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a portion of ActRIIB beginning at a residue correspondingto amino acids 20-29 (e.g., beginning at any one of amino acids 20, 21,22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 and ending at aposition corresponding to amino acids 109-134 (e.g., ending at any oneof amino acids 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or134) of SEQ ID NO: 1. In certain preferred embodiments, heteromultimersof the disclosure comprise at least one ActRIIB polypeptide thatcomprises, consists, or consists essentially of an amino acid sequencethat is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acids 29-109of SEQ ID NO: 1. In other preferred embodiments, heteromultimercomplexes of the disclosure comprise at least one ActRIIB polypeptidethat comprises, consists of, or consists essentially of an an amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical aminoacids 25-131 of SEQ ID NO: 1. In some embodiments, heteromultimers ofthe disclosure comprise at least one ActRIIB polypeptide that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of anyone of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 39, 41, 45, or 46. In certainpreferred embodiments, heteromultimers of the disclosure comprise do notcomprise an ActRIIB polypeptide wherein the position corresponding toL79 of SEQ ID NO: 1 is an acidic amino acid (i.e., is not a naturallyoccurring D or E amino acid residue or artificial acidic amino acid).

In certain aspects, the present disclosure relates to protein complexesthat comprise an ALK4 polypeptide. As used herein, the term “ALK4”refers to a family of activin receptor-like kinase-4 proteins from anyspecies and variants derived from such ALK4 proteins by mutagenesis orother modification. Reference to ALK4 herein is understood to be areference to any one of the currently identified forms. Members of theALK4 family are generally transmembrane proteins, composed of aligand-binding extracellular domain with a cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine kinase activity.

The term “ALK4 polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ALK4 family member as well as anyvariants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Numbering of aminoacids for all ALK4-related polypeptides described herein is based on thenumbering of the human ALK4 precursor protein sequence below (SEQ ID NO:9), unless specifically designated otherwise.

The canonical human ALK4 precursor protein sequence (NCBI Ref SeqNP_004293) is as follows:

(SEQ ID NO: 9)   1 MAESAGASSF FPLVVLLLAG SGGSGPRGVQ ALLCACTSCL QANYTCETDG ACMVSIFNLD 61 GMEHHVRTCI PKVELVPAGK PFYCLSSEDL RNTHCCYTDY CNRIDLRVPS GHLKEPEHPS121 MWGPVELVGI IAGPVFLLFL IIIIVFLVIN YHQRVYHNRQ RLDMEDPSCE MCLSKDKTLQ181 DLVYDLSTSG SGSGLPLFVQ RTVARTIVLQ EIIGKGRFGE VWRGRWRGGD VAVKIFSSRE241 ERSWFREAEI YQTVMLRHEN ILGFIAADNK DNGTWTQLWL VSDYHEHGSL FDYLNRYTVT301 IEGMIKLALS AASGLAHLHM EIVGTQGKPG IAHRDLKSKN ILVKKNGMCA IADLGLAVRH361 DAVTDTIDIA PNQRVGTKRY MAPEVLDETI NMKHFDSFKC ADIYALGLVY WEIARRCNSG421 GVHEEYQLPY YDLVPSDPSI EEMRKVVCDQ KLRPNIPNWW QSYEALRVMG KMMRECWYAN481 GAARLTALRI KKTLSQLSVQ EDVKI

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed (mature) extracellular human ALK4 polypeptide sequence isas follows:

(SEQ ID NO: 10) SGPRGVQALLCACTSCLQANYTCETDGACMVSIFNLDGMEHHVRTCIPKVELVPAGKPFYCLSSEDLRNTHCCYTDYCNRIDLRVPSGHLKEPEHP SMWGPVE

A nucleic acid sequence encoding the ALK4 precursor protein is shownbelow (SEQ ID NO: 11), corresponding to nucleotides 78-1592 of GenbankReference Sequence NM_004302.4. The signal sequence is underlined andthe extracellular domain is indicated in bold font.

(SEQ ID NO: 11) ATGGCGGAGTCGGCCGGAGCCTCCTCCTTCTTCCCCCTTGTTGTCCTCCTGCTCGCCGGCAGCGGCGGG TCCGGGCCCCGGGGGGTCCAGGCTCTGCTGTGTGCGTGCACCAGCTGCCTCCAGGCCAACTACACGTGTGAGACAGATGGGGCCTGCATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGCCCGGTGGAGCTGGTAGGCATCATCGCCGGCCCGGTGTTCCTCCTGTTCCTCATCATCATCATTGTTTTCCTTGTCATTAACTATCATCAGCGTGTCTATCACAACCGCCAGAGACTGGACATGGAAGATCCCTCATGTGAGATGTGTCTCTCCAAAGACAAGACGCTCCAGGATCTTGTCTACGATCTCTCCACCTCAGGGTCTGGCTCAGGGTTACCCCTCTTTGTCCAGCGCACAGTGGCCCGAACCATCGTTTTACAAGAGATTATTGGCAAGGGTCGGTTTGGGGAAGTATGGCGGGGCCGCTGGAGGGGTGGTGATGTGGCTGTGAAAATATTCTCTTCTCGTGAAGAACGGTCTTGGTTCAGGGAAGCAGAGATATACCAGACGGTCATGCTGCGCCATGAAAACATCCTTGGATTTATTGCTGCTGACAATAAAGATAATGGCACCTGGACACAGCTGTGGCTTGTTTCTGACTATCATGAGCACGGGTCCCTGTTTGATTATCTGAACCGGTACACAGTGACAATTGAGGGGATGATTAAGCTGGCCTTGTCTGCTGCTAGTGGGCTGGCACACCTGCACATGGAGATCGTGGGCACCCAAGGGAAGCCTGGAATTGCTCATCGAGACTTAAAGTCAAAGAACATTCTGGTGAAGAAAAATGGCATGTGTGCCATAGCAGACCTGGGCCTGGCTGTCCGTCATGATGCAGTCACTGACACCATTGACATTGCCCCGAATCAGAGGGTGGGGACCAAACGATACATGGCCCCTGAAGTACTTGATGAAACCATTAATATGAAACACTTTGACTCCTTTAAATGTGCTGATATTTATGCCCTCGGGCTTGTATATTGGGAGATTGCTCGAAGATGCAATTCTGGAGGAGTCCATGAAGAATATCAGCTGCCATATTACGACTTAGTGCCCTCTGACCCTTCCATTGAGGAAATGCGAAAGGTTGTATGTGATCAGAAGCTGCGTCCCAACATCCCCAACTGGTGGCAGAGTTATGAGGCACTGCGGGTGATGGGGAAGATGATGCGAGAGTGTTGGTATGCCAACGGCGCAGCCCGCCTGACGGCCCTGCGCATCAAGAAGACCCTCTCCCAGCTCAGCGTGCAG GAAGACGTGAAGATC 

A nucleic acid sequence encoding the extracellular ALK4 polypeptide isas follows:

(SEQ ID NO: 12) TCCGGGCCCCGGGGGGTCCAGGCTCTGCTGTGTGCGTGCACCAGCTGCCTCCAGGCCAACTACACGTGTGAGACAGATGGGGCCTGCATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGC CCGGTGGAG 

An alternative isoform of human ALK4 precursor protein sequence, isoformC (NCBI Ref Seq NP_064733.3), is as follows:

(SEQ ID NO: 19)   1 MAESAGASSF FPLVVLLLAG SGGSGPRGVQ ALLCACTSCL QANYTCETDG ACMVSIFNLD  61GMEHHVRTCI PKVELVPAGK PFYCLSSEDL RNTHCCYTDY CNRIDLRVPS GHLKEPEHPS 121MWGPVELVGI IAGPVFLLFL IIIIVFLVIN YHQRVYHNRQ RLDMEDPSCE MCLSKDKTLQ 181DLVYDLSTSG SGSGLPLFVQ RTVARTIVLQ EIIGKGRFGE VWRGRWRGGD VAVKIFSSRE 241ERSWFREAEI YQTVMLRHEN ILGFIAADNK ADCSFLTLPW EVVMVSAAPK LRSLRLQYKG 301GRGRARFLFP LNNGTWTQLW LVSDYHEHGS LFDYLNRYTV TIEGMIKLAL SAASGLAHLH 361MEIVGTQGKP GIAHRDLKSK NILVKKNGMC AIADLGLAVR HDAVTDTIDI APNQRVGTKR 421YMAPEVLDET INMKHFDSFK CADIYALGLV YWEIARRCNS GGVHEEYQLP YYDLVPSDPS 481IEEMRKVVCD QKLRPNIPNW WQSYEALRVM GKMMRECWYA NGAARLTALR IKKTLSQLSV 541QEDVKI

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed (mature) extracellular ALK4 polypeptide sequence (isoformC) is as follows:

(SEQ ID NO: 20) SGPRGVQALLCACTSCLQANYTCETDGACMVSIFNLDGMEHHVRTCIPKVELVPAGKPFYCLSSEDLRNTHCCYTDYCNRIDLRVPSGHLKEPEHPSMWG PVE 

A nucleic acid sequence encoding the ALK4 precursor protein (isoform C)is shown below (SEQ ID NO: 21), corresponding to nucleotides 78-1715 ofGenbank Reference Sequence NM_020328.3. The signal sequence isunderlined and the extracellular domain is indicated in bold font.

(SEQ ID NO: 21) ATGGCGGAGTCGGCCGGAGCCTCCTCCTTCTTCCCCCTTGTTGTCCTCCTGCTCGCCGGCAGCGGCGGG TCCGGGCCCCGGGGGGTCCAGGCTCTGCTGTGTGCGTGCACCAGCTGCCTCCAGGCCAACTACACGTGTGAGACAGATGGGGCCTGCATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGCCCGGTGGAGCTGGTAGGCATCATCGCCGGCCCGGTGTTCCTCCTGTTCCTCATCATCATCATTGTTTTCCTTGTCATTAACTATCATCAGCGTGTCTATCACAACCGCCAGAGACTGGACATGGAAGATCCCTCATGTGAGATGTGTCTCTCCAAAGACAAGACGCTCCAGGATCTTGTCTACGATCTCTCCACCTCAGGGTCTGGCTCAGGGTTACCCCTCTTTGTCCAGCGCACAGTGGCCCGAACCATCGTTTTACAAGAGATTATTGGCAAGGGTCGGTTTGGGGAAGTATGGCGGGGCCGCTGGAGGGGTGGTGATGTGGCTGTGAAAATATTCTCTTCTCGTGAAGAACGGTCTTGGTTCAGGGAAGCAGAGATATACCAGACGGTCATGCTGCGCCATGAAAACATCCTTGGATTTATTGCTGCTGACAATAAAGCAGACTGCTCATTCCTCACATTGCCATGGGAAGTTGTAATGGTCTCTGCTGCCCCCAAGCTGAGGAGCCTTAGACTCCAATACAAGGGAGGAAGGGGAAGAGCAAGATTTTTATTCCCACTGAATAATGGCACCTGGACACAGCTGTGGCTTGTTTCTGACTATCATGAGCACGGGTCCCTGTTTGATTATCTGAACCGGTACACAGTGACAATTGAGGGGATGATTAAGCTGGCCTTGTCTGCTGCTAGTGGGCTGGCACACCTGCACATGGAGATCGTGGGCACCCAAGGGAAGCCTGGAATTGCTCATCGAGACTTAAAGTCAAAGAACATTCTGGTGAAGAAAAATGGCATGTGTGCCATAGCAGACCTGGGCCTGGCTGTCCGTCATGATGCAGTCACTGACACCATTGACATTGCCCCGAATCAGAGGGTGGGGACCAAACGATACATGGCCCCTGAAGTACTTGATGAAACCATTAATATGAAACACTTTGACTCCTTTAAATGTGCTGATATTTATGCCCTCGGGCTTGTATATTGGGAGATTGCTCGAAGATGCAATTCTGGAGGAGTCCATGAAGAATATCAGCTGCCATATTACGACTTAGTGCCCTCTGACCCTTCCATTGAGGAAATGCGAAAGGTTGTATGTGATCAGAAGCTGCGTCCCAACATCCCCAACTGGTGGCAGAGTTATGAGGCACTGCGGGTGATGGGGAAGATGATGCGAGAGTGTTGGTATGCCAACGGCGCAGCCCGCCTGACGGCCCTGCGCATCAAGAAGACCCTCTCCCAGCTCAGCGTGCAGGAAGACGTGAAGATC 

A nucleic acid sequence encoding the extracellular ALK4 polypeptide(isoform C) is as follows:

(SEQ ID NO: 22) TCCGGGCCCCGGGGGGTCCAGGCTCTGCTGTGTGCGTGCACCAGCTGCCTCCAGGCCAACTACACGTGTGAGACAGATGGGGCCTGCATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGC CCGGTGGAG

In certain embodiments, the disclosure relates to heteromultimers thatcomprise at least one ALK4 polypeptide, which includes fragments,functional variants, and modified forms thereof. Preferably, ALK4polypeptides for use in accordance with inventions of the disclosure(e.g., heteromultimers comprising an ALK4 polypeptide and uses thereof)are soluble (e.g., an extracellular domain of ALK4). In other preferredembodiments, ALK4 polypeptides for use in accordance with the inventionsof the disclosure bind to and/or inhibit (antagonize) activity (e.g.,Smad signaling) of one or more TGF-beta superfamily ligands. In someembodiments, heteromultimers of the disclosure comprise at least oneALK4 polypeptide that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% identical to theamino acid sequence of SEQ ID NO: 9, 10, 19, 20, 42, 44, 47, or 48. Insome embodiments, heteromultimer complexes of the disclosure consist orconsist essentially of at least one ALK4 polypeptide that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 9,10, 19, 20, 42, 44, 47, or 48.

ALK4 is well-conserved among vertebrates, with large stretches of theextracellular domain completely conserved. For example, FIG. 7 depicts amulti-sequence alignment of a human ALK4 extracellular domain comparedto various ALK4 orthologs. Many of the ligands that bind to ALK4 arealso highly conserved. Accordingly, from these alignments, it ispossible to predict key amino acid positions within the ligand-bindingdomain that are important for normal ALK4-ligand binding activities aswell as to predict amino acid positions that are likely to be tolerantto substitution without significantly altering normal ALK4-ligandbinding activities. Therefore, an active, human ALK4 variant polypeptideuseful in accordance with the presently disclosed methods may includeone or more amino acids at corresponding positions from the sequence ofanother vertebrate ALK4, or may include a residue that is similar tothat in the human or other vertebrate sequences. Without meaning to belimiting, the following examples illustrate this approach to defining anactive ALK4 variant. V6 in the human ALK4 extracellular domain (SEQ IDNO: 59) is isoleucine in Mus muculus ALK4 (SEQ ID NO: 63), and so theposition may be altered, and optionally may be altered to anotherhydrophobic residue such as L, I, or F, or a non-polar residue such asA, as is observed in Gallus gallus ALK4 (SEQ ID NO: 62). E40 in thehuman extracellular domain is K in Gallus gallus ALK4, indicating thatthis site may be tolerant of a wide variety of changes, including polarresidues, such as E, D, K, R, H, S, T, P, G, Y, and probably a non-polarresidue such as A. S15 in the human extracellular domain is D in Gallusgallus ALK4, indicating that a wide structural variation is tolerated atthis position, with polar residues favored, such as S, T, R, E, K, H, G,P, G and Y. E40 in the human extracellular domain is K in Gallus gallusALK4, indicating that charged residues will be tolerated at thisposition, including D, R, K, H, as well as Q and N. R80 in the humanextracellular domain is K in Condylura cristata ALK4 (SEQ ID NO: 60),indicating that basic residues are tolerated at this position, includingR, K, and H. Y77 in the human extracellular domain is F in Sus scrofaALK4 (SEQ ID NO: 64), indicating that aromatic residues are tolerated atthis position, including F, W, and Y. P93 in the human extracellulardomain is relatively poorly conserved, appearing as S in Erinaceuseuropaeus ALK4 (SEQ ID NO: 61) and N in Gallus gallus ALK4, thusessentially any amino acid should be tolerated at this position.

Moreover, ALK4 proteins have been characterized in the art in terms ofstructural and functional characteristics, particularly with respect toligand binding [e.g., Harrison et al. (2003) J Biol Chem278(23):21129-21135; Romano et al. (2012) J Mol Model 18(8):3617-3625;and Calvanese et al. (2009) 15(3):175-183]. In addition to the teachingsherein, these references provide amply guidance for how to generate ALK4variants that retain one or more normal activities (e.g., ligand-bindingactivity).

For example, a defining structural motif known as a three-finger toxinfold is important for ligand binding by type I and type II receptors andis formed by conserved cysteine residues located at varying positionswithin the extracellular domain of each monomeric receptor [Greenwald etal. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett586:1860-1870]. Accordingly, the core ligand-binding domains of humanALK4, as demarcated by the outermost of these conserved cysteines,corresponds to positions 34-101 of SEQ ID NO: 9 (ALK4 precursor). Thus,the structurally less-ordered amino acids flanking thesecysteine-demarcated core sequences can be truncated by 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, residues at the N-terminus or 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26residues at the C-terminus without necessarily altering ligand binding.Exemplary ALK4 extracellular domains for N-terminal and/or C-terminaltruncation include SEQ ID NOs: 10 and 20.

Accordingly, a general formula for an active portion (e.g., aligand-binding portion) of ALK4 comprises amino acids 34-101. ThereforeALK4 polypeptides may, for example, comprise, consists essentially of,or consists of an amino acid sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to a portion of ALK4 beginning at a residuecorresponding to any one of amino acids 24-34 (e.g., beginning at anyone of amino acids 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34) of SEQID NO: 1 and ending at a position corresponding to any one amino acids101-126 (e.g., ending at any one of amino acids 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, or 126) of SEQ ID NO: 9. Other examplesinclude constructs that begin at a position from 24-34 (e.g., any one ofpositions 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34), 25-34 (e.g.,any one of positions 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34), or26-34 (e.g., any one of positions 26, 27, 28, 29, 30, 31, 32, 33, or 34)of SEQ ID NO: 9 and end at a position from 101-126 (e.g., any one ofpositions 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or126), 102-126 (e.g., any one of positions 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, or 126), 101-125 (e.g., any one of positions 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, or 125), 101-124 (e.g., anyone of positions 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124),101-121 (e.g., any one of positions 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or121), 111-126 (e.g., any one of positions 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, or 126), 111-125 (e.g., anyone of positions 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, or 125), 111-124 (e.g., any one of positions 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124), 121-126(e.g., any one of positions 121, 122, 123, 124, 125, or 126), 121-125(e.g., any one of positions 121, 122, 123, 124, or 125), 121-124 (e.g.,any one of positions 121, 122, 123, or 124), or 124-126 (e.g., any oneof positions 124, 125, or 126) of SEQ ID NO: 9. Variants within theseranges are also contemplated, particularly those having at least 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identity to the corresponding portion of SEQ IDNO: 9.

The variations described herein may be combined in various ways. In someembodiments, ALK4 variants comprise no more than 1, 2, 5, 6, 7, 8, 9, 10or 15 conservative amino acid changes in the ligand-binding pocket.Sites outside the binding pocket, at which variability may beparticularly well tolerated, include the amino and carboxy termini ofthe extracellular domain (as noted above),

In certain aspects, the present disclosure relates to heteromultimerscomprising one or more ALK4 receptor polypeptides (e.g., SEQ ID Nos: 9,10, 19, 20, 42, 44, 47 and 48) and one or more ActRIIB receptorpolypeptides (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 39, 41, 45, and 46),which are generally referred to herein as “ALK4:ActRIIB heteromultimercomplexes” or “ALK4:ActRIIB heteromultimers”. Preferably, ALK4:ActRIIBheteromultimers of the disclosure are soluble, e.g., a heteromultimerscomprises a soluble portion (domain) of an ALK4 receptor and a solubleportion (domain) of an ActRIIB receptor. In general, the extracellulardomains of ALK4 and ActRIIB correspond to soluble portion of thesereceptors. Therefore, in some embodiments, heteromultimers of thedisclosure comprise an extracellular domain of an ALK4, receptor and anextracellular domain of an ActRIIB receptor. Exemplary extracellulardomains ALK4 and ActRIIB receptors are disclosed herein and suchsequences, as well as fragments, functional variants, and modified formsthereof, may be used in accordance with the inventions of the disclosure(e.g., ALK4:ActRIIB heteromultimer compositions and uses thereof). Insome embodiments, ALK4:ActRIIB heteromultimers of the disclosurecomprise at least one ALK4 polypeptide that comprises, consistsessentially of, or consists a sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 97%, 98%, 99%, or100% identical to the amino acid sequence of SEQ ID NO: 9, 10, 19, 20,42, 44, 47, and 48. In some embodiments, ALK4:ActRIIB heteromultimers ofthe disclosure comprise at least one ALK4 polypeptide that comprises,consists essentially of, consists of a sequence that is at least 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 97%,98%, 99%, or 100% identical to a portion of ALK4 beginning at a residuecorresponding to any one of amino acids 24-34, 25-34, or 26-34 andending at a position from 101-126, 102-126, 101-125, 101-124, 101-121,111-126, 111-125, 111-124, 121-126, 121-125, 121-124, or 124-126 of SEQID NO: 9. In some embodiments, ALK4-ActRIIB heteromultimers of thedisclosure comprise at least one ActRIIB polypeptide that comprises,consists essentially of, consists of a sequence that is at least 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 97%,98%, 99%, or 100% identical to the amino acid sequence of any one of SEQID NOs: 1, 2, 3, 4, 5, 6, 39, 41, 45, and 46. In some embodiments,ALK4:ActRIIB heteromultimers of the disclosure comprise at least oneActRIIB polypeptide that comprises, consists essentially of, consists ofa sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94% 95%, 97%, 98%, 99%, or 100% identical to a portion ofActRIIB beginning at a residue corresponding to any one of amino acids20-29, 20-24, 21-24, 22-25, or 21-29 and end at a position from 109-134,119-134, 119-133, 129-134, or 129-133 of SEQ ID NO: 1. In certainpreferred embodiments, ALK4:ActRIIB heteromultimers of the disclosurecomprise at least one ActRIIB polypeptide wherein the positioncorresponding to L79 of SEQ ID NO: 1 is not an acidic amino acid (i.e.,not a naturally occurring D or E amino acid residue or an artificiallyacidic amino acid). ALK4:ActRIIB heteromultimers of the disclosureinclude, e.g., heterodimers, heterotrimers, heterotetramers and higherorder oligomeric structures. See, e.g., FIGS. 1, 2, and 8-10. In certainpreferred embodiments, heteromultimer complexes of the disclosure areALK4:ActRIIB heterodimers.

In some embodiments, the present disclosure contemplates makingfunctional variants by modifying the structure of an ALK4 polypeptideand/or an ActRIIB polypeptide. Variants can be produced by amino acidsubstitution, deletion, addition, or combinations thereof. For instance,it is reasonable to expect that an isolated replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, a threoninewith a serine, or a similar replacement of an amino acid with astructurally related amino acid (e.g., conservative mutations) will nothave a major effect on the biological activity of the resultingmolecule. Conservative replacements are those that take place within afamily of amino acids that are related in their side chains. Whether achange in the amino acid sequence of a polypeptide of the disclosureresults in a functional homolog can be readily determined by assessingthe ability of the variant polypeptide to produce a response in cells ina fashion similar to the wild-type polypeptide, or to bind to one ormore TGF-beta superfamily ligands including, for example, BMP2, BMP2/7,BMP3, BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3,GDF5, GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1,TGF-β1, TGF-β2, TGF-β3, activin A, activin B, activin C, activin E,activin AB, activin AC, activin BC, activin AE, activin BE, nodal, glialcell-derived neurotrophic factor (GDNF), neurturin, artemin, persephin,MIS, and Lefty.

In some embodiments, the present disclosure contemplates makingfunctional variants by modifying the structure of an ALK4 and/or ActRIIBpolypeptide for such purposes as enhancing therapeutic efficacy orstability (e.g., increase shelf-life and/or resistance to proteolyticdegradation).

In some embodiments, the present disclosure contemplates specificmutations of an ALK4 polypeptide and/or an ActRIIB polypeptide so as toalter the glycosylation of the polypeptide. Such mutations may beselected so as to introduce or eliminate one or more glycosylationsites, such as O-linked or N-linked glycosylation sites.Asparagine-linked glycosylation recognition sites generally comprise atripeptide sequence, asparagine-X-threonine or asparagine-X-serine(where “X” is any amino acid) which is specifically recognized byappropriate cellular glycosylation enzymes. The alteration may also bemade by the addition of, or substitution by, one or more serine orthreonine residues to the sequence of the polypeptide (for O-linkedglycosylation sites). A variety of amino acid substitutions or deletionsat one or both of the first or third amino acid positions of aglycosylation recognition site (and/or amino acid deletion at the secondposition) results in non-glycosylation at the modified tripeptidesequence. Another means of increasing the number of carbohydratemoieties on a polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Depending on the coupling mode used, thesugar(s) may be attached to (a) arginine and histidine; (b) freecarboxyl groups; (c) free sulfhydryl groups such as those of cysteine;(d) free hydroxyl groups such as those of serine, threonine, orhydroxyproline; (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan; or (f) the amide group of glutamine. Removal ofone or more carbohydrate moieties present on a polypeptide may beaccomplished chemically and/or enzymatically. Chemical deglycosylationmay involve, for example, exposure of a polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. [Meth. Enzymol. (1987)138:350]. The sequence of a polypeptide may be adjusted, as appropriate,depending on the type of expression system used, as mammalian, yeast,insect, and plant cells may all introduce differing glycosylationpatterns that can be affected by the amino acid sequence of the peptide.In general, heteromeric complexes of the present disclosure for use inhumans may be expressed in a mammalian cell line that provides properglycosylation, such as HEK293 or CHO cell lines, although othermammalian expression cell lines are expected to be useful as well.

The present disclosure further contemplates a method of generatingmutants, particularly sets of combinatorial mutants of an ALK4 and/or anActRIIB polypeptide as well as truncation mutants. Pools ofcombinatorial mutants are especially useful for identifying functionallyactive (e.g., TGF-beta superfamily ligand binding) ALK4 and/or ActRIIBsequences. The purpose of screening such combinatorial libraries may beto generate, for example, polypeptides variants which have alteredproperties, such as altered pharmacokinetic or altered ligand binding. Avariety of screening assays are provided below, and such assays may beused to evaluate variants. For example, ALK4:ActRIIB complex variantsmay be screened for ability to bind to one or more TGF-beta superfamilyligands to prevent binding of a TGF-beta superfamily ligand to aTGF-beta superfamily receptor, and/or to interfere with signaling causedby an TGF-beta superfamily ligand.

The activity of a ALK4:ActRIIB heteromultimer may be tested, forexample, in a cell-based or in vivo assay. For example, the effect of anALK4:ActRIIB heteromultimer on the expression of genes or activity ofproteins involved in muscle production in a muscle cell may be assessed.This may, as needed, be performed in the presence of one or moreTGF-beta superfamily ligands, and cells may be transfected so as toproduce an ALK4:ActRIIB heteromultimer, and optionally, a TGF-betasuperfamily ligand. Likewise, an ALK4:ActRIIB heteromultimer may beadministered to a mouse or other animal, and one or more measurements,such as muscle formation and strength may be assessed usingart-recognized methods. Similarly, the activity of an ALK4:ActRIIBheteromultimer, or variants thereof, may be tested, for example, inosteoblasts, adipocytes, and/or neuronal cells for any effect on growthof these cells, for example, by the assays as described herein and thoseof common knowledge in the art. A SMAD-responsive reporter gene may beused in such cell lines to monitor effects on downstream signaling.

Combinatorial-derived variants can be generated which have increasedselectivity or generally increased potency relative to a referenceALK4:ActRIIB heteromultimer. Such variants, when expressed fromrecombinant DNA constructs, can be used in gene therapy protocols.Likewise, mutagenesis can give rise to variants which have intracellularhalf-lives dramatically different than the corresponding unmodifiedALK4:ActRIIB heteromultimer. For example, the altered protein can berendered either more stable or less stable to proteolytic degradation orother cellular processes which result in destruction, or otherwiseinactivation, of an unmodified polypeptide. Such variants, and the geneswhich encode them, can be utilized to alter polypeptide complex levelsby modulating the half-life of the polypeptide. For instance, a shorthalf-life can give rise to more transient biological effects and, whenpart of an inducible expression system, can allow tighter control ofrecombinant polypeptide complex levels within the cell. In an Fc fusionprotein, mutations may be made in the linker (if any) and/or the Fcportion to alter one or more activities of the ALK4:ActRIIBheteromultimer including, for example, immunogenicity, half-life, andsolubility.

A combinatorial library may be produced by way of a degenerate libraryof genes encoding a library of polypeptides which each include at leasta portion of potential ALK4 and/or ActRIIB sequences. For instance, amixture of synthetic oligonucleotides can be enzymatically ligated intogene sequences such that the degenerate set of potential ALK4 and/orActRIIB encoding nucleotide sequences are expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display).

There are many ways by which the library of potential homologs can begenerated from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be carried out in an automatic DNAsynthesizer, and the synthetic genes can then be ligated into anappropriate vector for expression. The synthesis of degenerateoligonucleotides is well known in the art [Narang, S A (1983)Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc. 3rdCleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura etal. (1984) Science 198:1056; and Ike et al. (1983) Nucleic Acid Res.11:477]. Such techniques have been employed in the directed evolution ofother proteins [Scott et al., (1990) Science 249:386-390; Roberts et al.(1992) PNAS USA 89:2429-2433; Devlin et al. (1990) Science 249: 404-406;Cwirla et al., (1990) PNAS USA 87: 6378-6382; as well as U.S. Pat. Nos.5,223,409, 5,198,346, and 5,096,815].

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, ALK4:ActRIIB heteromultimers can begenerated and isolated from a library by screening using, for example,alanine scanning mutagenesis [Ruf et al. (1994) Biochemistry33:1565-1572; Wang et al. (1994) J. Biol. Chem. 269:3095-3099; Balint etal. (1993) Gene 137:109-118; Grodberg et al. (1993) Eur. J. Biochem.218:597-601; Nagashima et al. (1993) J. Biol. Chem. 268:2888-2892;Lowman et al. (1991) Biochemistry 30:10832-10838; and Cunningham et al.(1989) Science 244:1081-1085], by linker scanning mutagenesis [Gustin etal. (1993) Virology 193:653-660; and Brown et al. (1992) Mol. Cell Biol.12:2644-2652; McKnight et al. (1982) Science 232:316], by saturationmutagenesis [Meyers et al., (1986) Science 232:613]; by PCR mutagenesis[Leung et al. (1989) Method Cell Mol Biol 1:11-19]; or by randommutagenesis, including chemical mutagenesis [Miller et al. (1992) AShort Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor,N.Y.; and Greener et al. (1994) Strategies in Mol Biol 7:32-34]. Linkerscanning mutagenesis, particularly in a combinatorial setting, is anattractive method for identifying truncated (bioactive) forms of ALK4and/or ActRIIB polypeptides.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of ALK4:ActRIIB heteromultimers. The mostwidely used techniques for screening large gene libraries typicallycomprise cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Preferredassays include TGF-beta superfamily ligand (e.g., BMP2, BMP2/7, BMP3,BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5,GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1, TGF-β1,TGF-β2, TGF-β3, activin A, activin B, activin C, activin E, activin AB,activin AC, activin BC, activin AE, activin BE, nodal, glialcell-derived neurotrophic factor (GDNF), neurturin, artemin, persephin,MIS, and Lefty) binding assays and/or TGF-beta ligand-mediated cellsignaling assays.

In certain embodiments, ALK4:ActRIIB heteromultimers may furthercomprise post-translational modifications in addition to any that arenaturally present in the ALK4 and/or ActRIIB polypeptide. Suchmodifications include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. As a result, ALK4:ActRIIB heteromultimers may comprisenon-amino acid elements, such as polyethylene glycols, lipids,polysaccharide or monosaccharide, and phosphates. Effects of suchnon-amino acid elements on the functionality of a heteromultimer complexmay be tested as described herein for other heteromultimer variants.When a polypeptide of the disclosure is produced in cells by cleaving anascent form of the polypeptide, post-translational processing may alsobe important for correct folding and/or function of the protein.Different cells (e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293)have specific cellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the ALK4 and/or ActRIIB polypeptide aswell as heteromultimers comprising the same.

In certain preferred embodiments, heteromultimers described hereincomprise at least one ALK4 polypeptide associated, covalently ornon-covalently, with at least one ActRIIB polypeptide. Preferably,polypeptides disclosed herein form heterodimeric complexes, althoughhigher order heteromultimeric complexes are also included such as, butnot limited to, heterotrimers, heterotetramers, and further oligomericstructures (see, e.g., FIGS. 1, 2, and 8-10). In some embodiments, ALK4and/or ActRIIB polypeptides comprise at least one multimerizationdomain. As disclosed herein, the term “multimerization domain” refers toan amino acid or sequence of amino acids that promote covalent ornon-covalent interaction between at least a first polypeptide and atleast a second polypeptide. Polypeptides disclosed herein may be joinedcovalently or non-covalently to a multimerization domain. Preferably, amultimerization domain promotes interaction between a first polypeptide(e.g., an ALK4 polypeptide) and a second polypeptide (e.g., an ActRIIBpolypeptide) to promote heteromultimer formation (e.g., heterodimerformation), and optionally hinders or otherwise disfavors homomultimerformation (e.g., homodimer formation), thereby increasing the yield ofdesired heteromultimer (see, e.g., FIG. 2).

Many methods known in the art can be used to generate ALK4:ActRIIBheteromultimers. For example, non-naturally occurring disulfide bondsmay be constructed by replacing on a first polypeptide (e.g., an ALK4polypeptide) a naturally occurring amino acid with a freethiol-containing residue, such as cysteine, such that the free thiolinteracts with another free thiol-containing residue on a secondpolypeptide (e.g., an ActRIIB polypeptide) such that a disulfide bond isformed between the first and second polypeptides. Additional examples ofinteractions to promote heteromultimer formation include, but are notlimited to, ionic interactions such as described in Kjaergaard et al.,WO2007147901; electrostatic steering effects such as described in Kannanet al., U.S. Pat. No. 8,592,562; coiled-coil interactions such asdescribed in Christensen et al., U.S. 20120302737; leucine zippers suchas described in Pack & Plueckthun, (1992) Biochemistry 31: 1579-1584;and helix-turn-helix motifs such as described in Pack et al., (1993)Bio/Technology 11: 1271-1277. Linkage of the various segments may beobtained via, e.g., covalent binding such as by chemical cross-linking,peptide linkers, disulfide bridges, etc., or affinity interactions suchas by avidin-biotin or leucine zipper technology.

In certain aspects, a multimerization domain may comprise one componentof an interaction pair. In some embodiments, the polypeptides disclosedherein may form protein complexes comprising a first polypeptidecovalently or non-covalently associated with a second polypeptide,wherein the first polypeptide comprises the amino acid sequence of anALK4 polypeptide and the amino acid sequence of a first member of aninteraction pair; and the second polypeptide comprises the amino acidsequence of an ActRIIB polypeptide and the amino acid sequence of asecond member of an interaction pair. The interaction pair may be anytwo polypeptide sequences that interact to form a complex, particularlya heterodimeric complex although operative embodiments may also employan interaction pair that can form a homodimeric complex. One member ofthe interaction pair may be fused to an ALK4 or ActRIIB polypeptide asdescribed herein, including for example, a polypeptide sequencecomprising, consisting essentially of, or consisting of an amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence of any one of SEQ ID NOs: 2, 3, 5, 6, 10, and 20. Aninteraction pair may be selected to confer an improved property/activitysuch as increased serum half-life, or to act as an adaptor on to whichanother moiety is attached to provide an improved property/activity. Forexample, a polyethylene glycol moiety may be attached to one or bothcomponents of an interaction pair to provide an improvedproperty/activity such as improved serum half-life.

The first and second members of the interaction pair may be anasymmetric pair, meaning that the members of the pair preferentiallyassociate with each other rather than self-associate. Accordingly, firstand second members of an asymmetric interaction pair may associate toform a heterodimeric complex (see, e.g., FIG. 2). Alternatively, theinteraction pair may be unguided, meaning that the members of the pairmay associate with each other or self-associate without substantialpreference and thus may have the same or different amino acid sequences.Accordingly, first and second members of an unguided interaction pairmay associate to form a homodimer complex or a heterodimeric complex.Optionally, the first member of the interaction pair (e.g., anasymmetric pair or an unguided interaction pair) associates covalentlywith the second member of the interaction pair. Optionally, the firstmember of the interaction pair (e.g., an asymmetric pair or an unguidedinteraction pair) associates non-covalently with the second member ofthe interaction pair.

As specific examples, the present disclosure provides fusion proteinscomprising ALK4 or ActRIIB fused to a polypeptide comprising a constantdomain of an immunoglobulin, such as a CH1, CH2, or CH3 domain of animmunoglobulin or an Fc domain. Fc domains derived from human IgG1,IgG2, IgG3, and IgG4 are provided herein. Other mutations are known thatdecrease either CDC or ADCC activity, and collectively, any of thesevariants are included in the disclosure and may be used as advantageouscomponents of a heteromultimeric complex of the disclosure. Optionally,the IgG1 Fc domain of SEQ ID NO: 31 has one or more mutations atresidues such as Asp-265, Lys-322, and Asn-434 (numbered in accordancewith the corresponding full-length IgG1). In certain cases, the mutantFc domain having one or more of these mutations (e.g., Asp-265 mutation)has reduced ability of binding to the Fcγ receptor relative to awildtype Fc domain. In other cases, the mutant Fc domain having one ormore of these mutations (e.g., Asn-434 mutation) has increased abilityof binding to the MHC class I-related Fc-receptor (FcRN) relative to awildtype Fc domain.

An example of a native amino acid sequence that may be used for the Fcportion of human IgG1 (G1Fc) is shown below (SEQ ID NO: 31). Dottedunderline indicates the hinge region, and solid underline indicatespositions with naturally occurring variants. In part, the disclosureprovides polypeptides comprising, consisting essentially of, orconsisting of amino acid sequences with 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity to SEQ ID NO: 31. Naturally occurring variants in G1Fc wouldinclude E134D and M136L according to the numbering system used in SEQ IDNO: 31 (see Uniprot P01857).

(SEQ ID NO: 31)

An example of a native amino acid sequence that may be used for the Fcportion of human IgG2 (G2Fc) is shown below (SEQ ID NO: 32). Dottedunderline indicates the hinge region and double underline indicatespositions where there are data base conflicts in the sequence (accordingto UniProt P01859). In part, the disclosure provides polypeptidescomprising, consisting essential of, or consisting of amino acidsequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 32.

(SEQ ID NO: 32)

Two examples of amino acid sequences that may be used for the Fc portionof human IgG3 (G3Fc) are shown below. The hinge region in G3Fc can be upto four times as long as in other Fc chains and contains three identical15-residue segments preceded by a similar 17-residue segment. The firstG3Fc sequence shown below (SEQ ID NO: 33) contains a short hinge regionconsisting of a single 15-residue segment, whereas the second G3Fcsequence (SEQ ID NO: 34) contains a full-length hinge region. In eachcase, dotted underline indicates the hinge region, and solid underlineindicates positions with naturally occurring variants according toUniProt P01859. In part, the disclosure provides polypeptidescomprising, consisting essential of, or consisting of amino acidsequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 33and 34.

(SEQ ID NO: 33)

(SEQ ID NO: 34)

Naturally occurring variants in G3Fc (for example, see Uniprot P01860)include E68Q, P76L, E79Q, Y81F, D97N, N100D, T124A, S169N, S169de1,F221Y when converted to the numbering system used in SEQ ID NO: 33, andthe present disclosure provides fusion proteins comprising G3Fc domainscontaining one or more of these variations. In addition, the humanimmunoglobulin IgG3 gene (IGHG3) shows a structural polymorphismcharacterized by different hinge lengths [see Uniprot P01859].Specifically, variant WIS is lacking most of the V region and all of theCH1 region. It has an extra interchain disulfide bond at position 7 inaddition to the 11 normally present in the hinge region. Variant ZUClacks most of the V region, all of the CH1 region, and part of thehinge. Variant OMM may represent an allelic form or another gamma chainsubclass. The present disclosure provides additional fusion proteinscomprising G3Fc domains containing one or more of these variants.

An example of a native amino acid sequence that may be used for the Fcportion of human IgG4 (G4Fc) is shown below (SEQ ID NO: 35). Dottedunderline indicates the hinge region. In part, the disclosure providespolypeptides comprising, consisting essential of, or consisting of aminoacid sequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:35.

(SEQ ID NO: 35)

A variety of engineered mutations in the Fc domain are presented hereinwith respect to the G1Fc sequence (SEQ ID NO: 31), and analogousmutations in G2Fc, G3Fc, and G4Fc can be derived from their alignmentwith G1Fc in FIG. 5. Due to unequal hinge lengths, analogous Fcpositions based on isotype alignment (FIG. 5) possess different aminoacid numbers in SEQ ID NOs: 31, 32, 33, 34, and 35. It can also beappreciated that a given amino acid position in an immunoglobulinsequence consisting of hinge, C_(H)2, and C_(H)3 regions (e.g., SEQ IDNOs: 31, 32, 33, 34, and 35) will be identified by a different numberthan the same position when numbering encompasses the entire IgG1heavy-chain constant domain (consisting of the C_(H)1, hinge, C_(H)2,and C_(H)3 regions) as in the Uniprot database. For example,correspondence between selected C_(H)3 positions in a human G1Fcsequence (SEQ ID NO: 31), the human IgG1 heavy chain constant domain(Uniprot P01857), and the human IgG1 heavy chain is as follows.

Correspondence of C_(H)3 Positions in Different Numbering Systems G1Fc(Numbering begins IgG1 heavy chain IgG1 heavy chain at first constantdomain (EU numbering threonine in (Numbering begins scheme of hingeregion) at C_(H)1) Kabat et al., 1991*) Y127 Y232 Y349 S132 S237 S354E134 E239 E356 T144 T249 T366 L146 L251 L368 K170 K275 K392 D177 D282D399 Y185 Y290 Y407 K187 K292 K409 *Kabat et al. (eds) 1991; pp. 688-696in Sequences of Proteins of Immunological Interest, 5^(th) ed., Vol. 1,NIH, Bethesda, MD.

A problem that arises in large-scale production of asymmetricimmunoglobulin-based proteins from a single cell line is known as the“chain association issue”. As confronted prominently in the productionof bispecific antibodies, the chain association issue concerns thechallenge of efficiently producing a desired multichain protein fromamong the multiple combinations that inherently result when differentheavy chains and/or light chains are produced in a single cell line[Klein et al (2012) mAbs 4:653-663]. This problem is most acute when twodifferent heavy chains and two different light chains are produced inthe same cell, in which case there are a total of 16 possible chaincombinations (although some of these are identical) when only one istypically desired. Nevertheless, the same principle accounts fordiminished yield of a desired multichain fusion protein thatincorporates only two different (asymmetric) heavy chains.

Various methods are known in the art that increase desired pairing ofFc-containing fusion polypeptide chains in a single cell line to producea preferred asymmetric fusion protein at acceptable yields [Klein et al(2012) mAbs 4:653-663; and Spiess et al (2015) Molecular Immunology67(2A): 95-106]. Methods to obtain desired pairing of Fc-containingchains include, but are not limited to, charge-based pairing(electrostatic steering), “knobs-into-holes” steric pairing, SEEDbodypairing, and leucine zipper-based pairing [Ridgway et al (1996) ProteinEng 9:617-621; Merchant et al (1998) Nat Biotech 16:677-681; Davis et al(2010) Protein Eng Des Sel 23:195-202; Gunasekaran et al (2010);285:19637-19646; Wranik et al (2012) J Biol Chem 287:43331-43339; U.S.Pat. No. 5,932,448; WO 1993/011162; WO 2009/089004, and WO 2011/034605].As described herein, these methods may be used to generateALK4-Fc:ActRIIB-Fc heteromultimer complexes. See FIGS. 8-10.

For example, one means by which interaction between specificpolypeptides may be promoted is by engineering protuberance-into-cavity(knob-into-holes) complementary regions such as described in Arathoon etal., U.S. Pat. No. 7,183,076 and Carter et al., U.S. Pat. No. 5,731,168.“Protuberances” are constructed by replacing small amino acid sidechains from the interface of the first polypeptide (e.g., a firstinteraction pair) with larger side chains (e.g., tyrosine ortryptophan). Complementary “cavities” of identical or similar size tothe protuberances are optionally created on the interface of the secondpolypeptide (e.g., a second interaction pair) by replacing large aminoacid side chains with smaller ones (e.g., alanine or threonine). Where asuitably positioned and dimensioned protuberance or cavity exists at theinterface of either the first or second polypeptide, it is onlynecessary to engineer a corresponding cavity or protuberance,respectively, at the adjacent interface.

At neutral pH (7.0), aspartic acid and glutamic acid are negativelycharged and lysine, arginine, and histidine are positively charged.These charged residues can be used to promote heterodimer formation andat the same time hinder homodimer formation. Attractive interactionstake place between opposite charges and repulsive interactions occurbetween like charges. In part, protein complexes disclosed herein makeuse of the attractive interactions for promoting heteromultimerformation (e.g., heterodimer formation), and optionally repulsiveinteractions for hindering homodimer formation (e.g., homodimerformation) by carrying out site directed mutagenesis of chargedinterface residues.

For example, the IgG1 CH3 domain interface comprises four unique chargeresidue pairs involved in domain-domain interactions: Asp356-Lys439′,Glu357-Lys370′, Lys392-Asp399′, and Asp399-Lys409′ [residue numbering inthe second chain is indicated by (′)]. It should be noted that thenumbering scheme used here to designate residues in the IgG1 CH3 domainconforms to the EU numbering scheme of Kabat. Due to the 2-fold symmetrypresent in the CH3-CH3 domain interactions, each unique interaction willrepresented twice in the structure (e.g., Asp-399-Lys409′ andLys409-Asp399′). In the wild-type sequence, K409-D399′ favors bothheterodimer and homodimer formation. A single mutation switching thecharge polarity (e.g., K409E; positive to negative charge) in the firstchain leads to unfavorable interactions for the formation of the firstchain homodimer. The unfavorable interactions arise due to the repulsiveinteractions occurring between the same charges (negative-negative;K409E-D399′ and D399-K409E′). A similar mutation switching the chargepolarity (D399K′; negative to positive) in the second chain leads tounfavorable interactions (K409′-D399K′ and D399K-K409′) for the secondchain homodimer formation. But, at the same time, these two mutations(K409E and D399K′) lead to favorable interactions (K409E-D399K′ andD399-K409′) for the heterodimer formation.

The electrostatic steering effect on heterodimer formation and homodimerdiscouragement can be further enhanced by mutation of additional chargeresidues which may or may not be paired with an oppositely chargedresidue in the second chain including, for example, Arg355 and Lys360.The table below lists possible charge change mutations that can be used,alone or in combination, to enhance ALK4:ActRIIB heteromultimerformation.

Examples of Pair-Wise Charged Residue Mutations to Enhance HeterodimerFormation Corresponding Position in Mutation in Interacting position inmutation in first chain first chain second chain second chain Lys409 Aspor Glu Asp399′ Lys, Arg, or His Lys392 Asp or Glu Asp399′ Lys, Arg, orHis Lys439 Asp or Glu Asp356′ Lys, Arg, or His Lys370 Asp or Glu Glu357′Lys, Arg, or His Asp399 Lys, Arg, or His Lys409′ Asp or Glu Asp399 Lys,Arg, or His Lys392′ Asp or Glu Asp356 Lys, Arg, or His Lys439′ Asp orGlu Glu357 Lys, Arg, or His Lys370′ Asp or Glu

In some embodiments, one or more residues that make up the CH3-CH3interface in a fusion protein of the instant application are replacedwith a charged amino acid such that the interaction becomeselectrostatically unfavorable. For example, a positive-charged aminoacid in the interface (e.g., a lysine, arginine, or histidine) isreplaced with a negatively charged amino acid (e.g., aspartic acid orglutamic acid). Alternatively, or in combination with the forgoingsubstitution, a negative-charged amino acid in the interface is replacedwith a positive-charged amino acid. In certain embodiments, the aminoacid is replaced with a non-naturally occurring amino acid having thedesired charge characteristic. It should be noted that mutatingnegatively charged residues (Asp or Glu) to His will lead to increase inside chain volume, which may cause steric issues. Furthermore, Hisproton donor- and acceptor-form depends on the localized environment.These issues should be taken into consideration with the designstrategy. Because the interface residues are highly conserved in humanand mouse IgG subclasses, electrostatic steering effects disclosedherein can be applied to human and mouse IgG1, IgG2, IgG3, and IgG4.This strategy can also be extended to modifying uncharged residues tocharged residues at the CH3 domain interface.

In part, the disclosure provides desired pairing of asymmetricFc-containing polypeptide chains using Fc sequences engineered to becomplementary on the basis of charge pairing (electrostatic steering).One of a pair of Fc sequences with electrostatic complementarity can bearbitrarily fused to the ALK4 or ActRIIB polypeptide of the construct,with or without an optional linker, to generate an ALK4:ActRIIBheteromultimer. This single chain can be coexpressed in a cell of choicealong with the Fc sequence complementary to the first Fc to favorgeneration of the desired multichain construct (e.g., ALK4:ActRIIBheteromultimer). In this example based on electrostatic steering, SEQ IDNO: 23 [human G1Fc(E134K/D177K)] and SEQ ID NO: 24 [humanG1Fc(K170D/K187D)] are examples of complementary Fc sequences in whichthe engineered amino acid substitutions are double underlined, and theTGF-beta superfamily type I or type II receptor polypeptide of theconstruct can be fused to either SEQ ID NO: 23 or SEQ ID NO: 24, but notboth. Given the high degree of amino acid sequence identity betweennative hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can beappreciated that amino acid substitutions at corresponding positions inhG2Fc, hG3Fc, or hG4Fc (see FIG. 5) will generate complementary Fc pairswhich may be used instead of the complementary hG1Fc pair below (SEQ IDNOs: 23 and 24).

(SEQ ID NO: 23)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSRKEMTKNQ VSLTCLVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLKSDG SFFLYSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGK (SEQ ID NO: 24)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151YPSDIAVEWE SNGQPENNYD TTPPVLDSDG SFFLYSDLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGK

In part, the disclosure provides desired pairing of asymmetricFc-containing polypeptide chains using Fc sequences engineered forsteric complementarity. In part, the disclosure providesknobs-into-holes pairing as an example of steric complementarity. One ofa pair of Fc sequences with steric complementarity can be arbitrarilyfused to the ALK4 or ActRIIB polypeptide of the construct, with orwithout an optional linker, to generate an ALK4:ActRIIB heteromultimer.This single chain can be co-expressed in a cell of choice along with theFc sequence complementary to the first Fc to favor generation of thedesired multi-chain construct. In this example based on knobs-into-holespairing, SEQ ID NO: 25 [human G1Fc(T144Y)] and SEQ ID NO: 26 [humanG1Fc(Y185T)] are examples of complementary Fc sequences in which theengineered amino acid substitutions are double underlined, and the ALK4or ActRIIB polypeptide of the construct can be fused to either SEQ IDNO: 25 or SEQ ID NO: 26, but not both. Given the high degree of aminoacid sequence identity between native hG1Fc, native hG2Fc, native hG3Fc,and native hG4Fc, it can be appreciated that amino acid substitutions atcorresponding positions in hG2Fc, hG3Fc, or hG4Fc (see FIG. 5) willgenerate complementary Fc pairs which may be used instead of thecomplementary hG1Fc pair below (SEQ ID NOs: 25 and 26).

(SEQ ID NO: 25)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLYCLVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGK (SEQ ID NO: 26)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLTSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGK

An example of Fc complementarity based on knobs-into-holes pairingcombined with an engineered disulfide bond is disclosed in SEQ ID NO: 27[hG1Fc(S132C/T144W)] and SEQ ID NO: 28 [hG1Fc(Y127C/T144S/L146A/Y185V)].The engineered amino acid substitutions in these sequences are doubleunderlined, and the TGF-beta superfamily type I or type II polypeptideof the construct can be fused to either SEQ ID NO: 27 or SEQ ID NO: 28,but not both. Given the high degree of amino acid sequence identitybetween native hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, itcan be appreciated that amino acid substitutions at correspondingpositions in hG2Fc, hG3Fc, or hG4Fc (see FIG. 5) will generatecomplementary Fc pairs which may be used instead of the complementaryhG1Fc pair below (SEQ ID NOs: 27 and 28).

(SEQ ID NO: 27)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PCREEMTKNQ VSLWCLVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGK (SEQ ID NO: 28)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVCTLP PSREEMTKNQ VSLSCAVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLVSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGK

In part, the disclosure provides desired pairing of asymmetricFc-containing polypeptide chains using Fc sequences engineered togenerate interdigitating β-strand segments of human IgG and IgA C_(H)3domains. Such methods include the use of strand-exchange engineereddomain (SEED) C_(H)3 heterodimers allowing the formation of SEEDbodyfusion proteins [Davis et al. (2010) Protein Eng Design Sel 23:195-202].One of a pair of Fc sequences with SEEDbody complementarity can bearbitrarily fused to the ALK4 or ActIIB of the construct, with orwithout an optional linker, to generate a ALK4 or ActRIIB fusionpolypeptide. This single chain can be co-expressed in a cell of choicealong with the Fc sequence complementary to the first Fc to favorgeneration of the desired multi-chain construct. In this example basedon SEEDbody (Sb) pairing, SEQ ID NO: 29 [hG1Fc(Sb_(AG))] and SEQ ID NO:30 [hG1Fc(Sb_(GA))] are examples of complementary IgG Fc sequences inwhich the engineered amino acid substitutions from IgA Fc are doubleunderlined, and the ALK4 or ActRIIB polypeptide of the construct can befused to either SEQ ID NO: 29 or SEQ ID NO: 30, but not both. Given thehigh degree of amino acid sequence identity between native hG1Fc, nativehG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that aminoacid substitutions at corresponding positions in hG1Fc, hG2Fc, hG3Fc, orhG4Fc (see FIG. 5) will generate an Fc monomer which may be used in thecomplementary IgG-IgA pair below (SEQ ID NOs: 29 and 30).

(SEQ ID NO: 29)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PFRPEVHLLP PSREEMTKNQ VSLTCLARGF 151YPKDIAVEWE SNGQPENNYK TIPSRQEPSQ GTTTFAVTSK LTVDKSRWQQ 201GNVFSCSVMH EALHNHYTQK TISLSPGK (SEQ ID NO: 30)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PPSEELALNE LVTLTCLVKG 151FYPSDIAVEW ESNGQELPRE KYLTWAPVLD SDGSFFLYSI LRVAAE DWKK 201GDTFSCSVMH EALHNHYTQK SLDRSPGK

In part, the disclosure provides desired pairing of asymmetricFc-containing polypeptide chains with a cleavable leucine zipper domainattached at the C-terminus of the Fc C_(H)3 domains. Attachment of aleucine zipper is sufficient to cause preferential assembly ofheterodimeric antibody heavy chains [Wranik et al (2012) J Biol Chem287:43331-43339]. As disclosed herein, one of a pair of Fc sequencesattached to a leucine zipper-forming strand can be arbitrarily fused tothe ALK4 or ActRIIB polypeptide of the construct, with or without anoptional linker, to generate a ALK4 or ActRIIB fusion polypeptide. Thissingle chain can be co-expressed in a cell of choice along with the Fcsequence attached to a complementary leucine zipper-forming strand tofavor generation of the desired multi-chain construct. Proteolyticdigestion of the construct with the bacterial endoproteinase Lys-C postpurification can release the leucine zipper domain, resulting in an Fcconstruct whose structure is identical to that of native Fc. In thisexample based on leucine zipper pairing, SEQ ID NO: 36 [hG1Fc-Ap1(acidic)] and SEQ ID NO: 37 [hG1Fc-Bp1 (basic)] are examples ofcomplementary IgG Fc sequences in which the engineered complimentaryleucine zipper sequences are underlined, and the ALK4 or ActRIIBpolypeptide of the construct can be fused to either SEQ ID NO: 36 or SEQID NO: 37, but not both. Given the high degree of amino acid sequenceidentity between native hG1Fc, native hG2Fc, native hG3Fc, and nativehG4Fc, it can be appreciated that leucine zipper-forming sequencesattached, with or without an optional linker, to hG1Fc, hG2Fc, hG3Fc, orhG4Fc (see FIG. 5) will generate an Fc monomer which may be used in thecomplementary leucine zipper-forming pair below (SEQ ID NOs: 36 and 37).

(SEQ ID NO: 36)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGKGGSAQ LEKELQALEK ENAQLEWELQ 251 ALEKELAQGA T(SEQ ID NO: 37)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGKGGSAQ LKKKLQALKK KNAQLKWKLQ 251 ALKKKLAQGA T

As described above, various methods are known in the art that increasedesired pairing of Fc-containing fusion polypeptide chains in a singlecell line to produce a preferred asymmetric fusion protein at acceptableyields [Klein et al (2012) mAbs 4:653-663; and Spiess et al (2015)Molecular Immunology 67(2A): 95-106]. In addition, ALK4:ActRIIBheteromultimers may be generated using a combination of heavy and lightchain fusion proteins comprising either an ALK4 or ActRIIB polypeptide.For example, in some embodiments, an ALK4 polypeptide may be fused, withor without a linker domain, to an immunoglobulin heavy chain (IgG1,IgG2, IgG3, IgG4, IgM, IgA1, or IgA2) that comprises at least a portionof the C_(H)1 domain. Similarly, an ActRIIB polypeptide may be fused,with or without a linker domain, to an immunoglobulin light chain (kappaor lambda) that comprises at least a portion of the light chain constantdomain (C_(L)). In alternative embodiments, an ActRIIB polypeptide maybe fused, with or without a linker domain, to an immunoglobulin heavychain (IgG1, IgG2, IgG3, IgG4, IgM, IgA1, or IgA2) that comprises atleast a portion of the C_(H)1 domain, and an ALK4 polypeptide may befused, with or without a linker domain, to an immunoglobulin light chain(kappa or lambda) that comprises at least a portion of the light chainconstant domain (C_(L)). This design takes advantage of the naturalability of the heavy chains to heterodimerize with light chains. Inparticular, heterodimerization of a heavy and light chain occurs betweenthe C_(H)1 with the C_(L), which is generally stabilized by covalentlinking of the two domains via a disulfide bridge. Constructs employingthe full-length heavy chain, or at least a portion of the heavy chaincomprising the hinge region, could give rise to antibody-like moleculescomprising two “light chains” and two “heavy chains”. See FIG. 9. Apotential advantage of this design is that it may more closely mimic thenaturally occurring ALK4-ligand-ActRIIB complex and may display higheraffinity for the ligand than comparable single heterodimers. In someembodiments, this design may be modified by incorporating various heavychain truncations including, for example, truncations that comprise theC_(H)1 domain and some or all of the hinge domain (giving rise toF(ab′)₂-like molecules) as well as truncations that only comprise theC_(H)1 domain or a fragment thereof (giving rise to Fab-like molecules).See FIG. 9G. Various methods for designing such heteromultimerconstructs are described in US 2009/0010879, Klein et al [(2012) mAbs4:653-663], and Spiess et al [(2015) Molecular Immunology 67(2A):95-106] the contents of which are incorporated in their entirety herein.

In some embodiments, it is desirable to generate antibody-likeALK4:ActRIIB heterodimers comprising at least one branch of the complexcomprising an ALK4-C_(L):ActRIIB-C_(H)1 heterodimer pair and at least asecond branch comprising an ActRIIB-C_(L):ALK4-C_(H)1 heterodimer pair.See, e.g., FIG. 9B. Such heterodimer complexes can be generated, forexample, using combinations of heavy chain and light chain asymmetricalpairing technologies [Spiess et al (2015) Molecular Immunology 67(2A):95-106]. For example, in CrossMab technology, [Schaefer et al (2011).Proc. Natl. Acad. Sci. U.S.A. 108: 11187-11192] light chain mispairingis overcome using domain crossovers and heavy chains heterodimerizedusing knobs-into-holes [Merchant et al (1998) Nat. Biotechnol. 16:677-681}. For the domain crossovers either the variable domains or theconstant domains are swapped between light and heavy chains to createtwo asymmetric Fab arms that drive cognate light chain pairing whilepreserving the structural and functional integrity of the variabledomain [Fenn et al (2013) PLoS ONE 8: e61953]. An alternative approachfor overcoming light chain mispairing is designing heavy and lightchains with orthogonal Fab inter-faces [Lewis (2014) Nat. Biotechnol.32: 191-198]. This has been accomplished by computational modeling [Daset al (2008) Annu. Rev. Biochem. 77: 363-382] in combination with X-raycrystallography to identify mutations at the V_(H)/V_(L) andC_(H)1/C_(L) interfaces. For the heterodimers generated using thismethodology, it may be necessary to engineer mutations into bothV_(H)/V_(L) and C_(H)1/C_(L) interfaces to minimize heavy/light chainmispairing. The designed orthogonal Fab interface may be used inconjunction with a heavy chain heterodimerization strategy to facilitateefficient IgG production in a single host cell. Electrostatic steeringmay also be used to generate orthogonal Fab interfaces to facilitate theconstruction of such heterodimers. Peptide linkers may be used to ensurecognate pairing of light and heavy chains in a format known as “LUZ-Y”[Wranik et al (2012) J. Biol. Chem. 287: 43331-43339], wherein heavychain heterodimerization is accomplished using leucine zippers which maybe subsequently removed by proteolysis in vitro.

Alternatively, ALK4:ActRIIB heteromultimers may comprise one or moresingle-chain ligand traps as described herein, optionally which may becovalently or non-covalently associated with one or more ALK4 or ActRIIBpolypeptides as well as additional ALK4:ActRIIB single chain ligandtraps [US 2011/0236309 and US2009/0010879]. See FIG. 12. As describedherein, single-chain ligand traps do not require fusion to anymultimerization domain such as coiled-coil Fc domains to be multivalent.In general, single-chain ligand traps of the present disclosure compriseat least one ALK4 polypeptide domain and one ActRIIB polypeptide domain.The ALK4 and ActRIIB polypeptide domains, generally referred to hereinas binding domains (BD), optionally may be joined by a linker region.

For example, in one aspect, the present disclosure providesheteromultimers comprising a polypeptide having the following structure:

(<BD1>-linker1)_(k)-[<BD2>-linker2-{<BD3>-linker3}_(f)]_(n)-(<BD4>)_(m)-(linker4-BD5>_(d))_(h)

where: n and h are independently greater than or equal to one; d, f, m,and k are independently equal to or greater than zero; BD1, BD2, BD3,BD4, and BD5 are independently ALK4 or ActRIIB polypeptide domains,wherein at least one of BD1, BD2, BD3, and BD4 is an ALK4 polypeptidedomain, and wherein at least one of BD1, BD2, BD3, and BD4 is an ActRIIBpolypeptide domain, and linker1, linker2, linker3, and linker 4 areindependently greater than or equal to zero. In some embodiment,ALK4:ActRIIB single-chain traps comprise at least two different ALK4polypeptides. In some embodiments, ALK4:ActRIIB single-chain trapscomprise at least two different ActRIIB polypeptides. In someembodiment, ALK4:ActRIIB single-chain traps comprise at least twodifferent linkers. Depending on the values of selected for d, f, h, k,m, and n, the heteromultimer structure may comprise a large number ofrepeating units in various combinations or may be a relatively simplestructure.

In another aspect, the present disclosure provides heteromultimerscomprising a polypeptide having the following structure:

<BD1>-linker1-<BD2>

In yet another aspect, the present disclosure provides heteromultimerscomprising a polypeptide having the following structure:

<BD1>-(linker2-<BD2>)_(n)

where n is greater than or equal one.

Another aspect of the invention provides heteromultimers comprising apolypeptide having the following structure:

(<BD1>-linker1-<BD1>)_(f)-linker2-(<BD2>-linker3-<BD3>)_(g)

wherein f and g are greater than or equal to one.

In an embodiment where BD2 and BD3 are the same, and f and g are thesame number, this can result in a substantially mirror symmetricstructure around linker 2, subject to differences in the linkers. Ininstances where BD2 is different from BD3 and/or where f and g aredifferent numbers, different structures will be produced. It is withinthe capacity of one of ordinary skill in the art to select suitablebinding domains, linkers, and repeat frequencies in light of thedisclosure herein and knowledge in the art. Specific, non-limitingexamples of such single-chain ligand traps in accordance with thepresent disclosure are represented schematically in FIG. 11.

The linkers (1, 2, 3, and 4) may be the same or different. The linkerregion provides a segment that is distinct from the structuredligand-binding domains of ALK4 and ActRIIB and thus can be used forconjugation to accessory molecules (e.g., molecules useful in increasingstability such as PEGylation moieties) without having to chemicallymodify the binding domains. The linker may include an unstructured aminoacid sequence that may be either the same as or derived fromconservative modifications to the sequence of a natural unstructuredregion in the extracellular portion of the receptor for the ligand ofinterest or another receptor in the TGF-β superfamily. In otherinstances, such linkers may be entirely artificial in composition andorigin but will contain amino acids selected to provide an unstructuredflexible linker with a low likelihood of encountering electrostatic orsteric hindrance complications when brought into close proximity to theligand of interest. Linker length will be considered acceptable when itpermits binding domains located on each of the N- and C-termini of thelinker to bind their natural binding sites on their natural ligand suchthat, with both binding domains so bound, the ligand is bound with ahigher affinity than it would be bound by binding of only one of thebinding domains. In some instances, the number of amino acid residues inthe linker of either natural or artificial origin is selected to beequal to or greater than the minimum required distance for simultaneous(bridged) binding to two binding sites on the ALK4 and/or ActRIIBligand. For example, and without wishing to be limiting in any manner,the linker length may be between 1-10 amino acids, 10-20 amino acids,about 18-80 amino acids, 25-60 amino acids, 35-45 amino acids, or anyother suitable length.

Linkers may be designed to facilitate purification of the polypeptide.The exact purification scheme chosen will determine what modificationsare needed, for example and without wishing to be limiting, additions ofpurification “tags” such as His tags is contemplated; in other examples,the linker may include regions to facilitate the addition of cargo oraccessory molecules. When such additions affect the unstructured natureof the linker or introduce potential electrostatic or steric concerns,appropriate increases to the linker length will be made to ensure thatthe two binding domains are able to bind their respective sites on theligand. In light of the methods and teachings herein, suchdeterminations could be made routinely by one skilled in the art.

In addition, the present design permits linkage of other cargo molecules(for example imaging agents like fluorescent molecules), toxins, etc.For example, and without wishing to be limiting in any manner,single-chain polypeptides can be modified to add one or more cargoand/or accessory molecules (referred to collectively herein by R1, R2,R3, R4, etc.):

Without limiting the generality of R substituents available, R1, R2, R3,R4, R5, R6, R7, R8, R9, may or may not be present; when present, theymay be the same or different, and may independently be one or more of: afusion protein for targeting, for example, but not limited to such as anantibody fragment (e.g. single chain Fv) and/or a single domain antibody(sdAb); a radiotherapy and/or imaging agent, for example, but notlimited to a radionuceotide (e.g. ¹²³I, ¹¹¹In, ¹⁸F, ⁶⁴C, ⁶⁸Y, ¹²⁴I,¹³¹I, ⁹⁰Y, ¹⁷⁷Lu, ⁵⁷Cu, ²¹³Bi, ²¹¹At), a fluorescent dye (e.g. AlexaFluor, Cy dye) and/or a fluorescent protein tag (e.g. GFP, DsRed); acytotoxic agent for chemotherapy, for example, but not limited todoxorubicin, calicheamicin, a maytansinoid derivatives (e.g. DM1, DM4),a toxin (eg. truncated Pseudomonas endotoxin A, diphteria toxin); ananoparticle-based carrier, for example, but not limited to polyethyleneglycol (PEG), a polymer-conjugated to drug, nanocarrier or imaging agent(e.g. of a polymer N-(2-hydorxylpropyl) methacrylamide (HPMA), glutamicacid, PEG, dextran); a drug (for example, but not limited todoxorubicin, camptothecin, paclitaxel, palatinate); a nanocarrier, forexample, but not limited to a nanoshell or liposome; an imaging agent,for example, but not limited to Supermagnetic Iron Oxide (SPIO); adendrimer; and/or a solid support for use in ligand purification,concentration or sequestration (e.g. nanoparticles, inert resins,suitable silica supports).

In general, it will not be preferable to have cargo or accessorymolecules in all possible positions, as this may cause steric orelectrostatic complications. However, the effects of adding a cargo oraccessory molecule to any given position or positions on the structurecan be determined routinely in light of the disclosure herein bymodeling the linker between the binding domains and carrying outmolecular dynamics simulations to substantially minimize molecularmechanics energy and reduce steric and electrostatic incompatibilitybetween the linker and the ALK4 and ActRIIB polypeptides as taughtherein.

It may be preferable to add the cargo or accessory molecule to thelinker portion of the agent, rather to the binding domain, to reduce thelikelihood of interference in binding function. However, addition to thebinding domain is possible and could be desirable in some instances andthe effect of such an addition can be determined routinely in advance bymodeling the binding agent and the linker with the proposed addition asdescribed herein.

Conjugation methodologies can be performed using commercial kits thatenable conjugation via common reactive groups such as primary amines,succinimidyl (NHS) esters and sulfhydral-reactive groups. Somenon-limiting examples are: Alexa Fluor 488 protein labeling kit(Molecular Probes, Invitrogen detection technologies) and PEGylationkits (Pierce Biotechnology Inc.).

In certain aspects, ALK4:ActRIIB single-chain traps may be covalently ornon-covalently associated with one or more ALK4 or ActRIIB polypeptidesas well as additional ALK4:ActRIIB single chain ligand traps to formhigher order heteromultimers, which may be used in accordance with themethods described herein. See, e.g., FIG. 12. For example, anALK4:ActRIIB single chain ligand trap may further comprise amultimerization domain as described herein. In some embodiments,ALK4:ActRIIB single chain ligand traps comprise a constant domain of anIg immunoglobulin. Such immunoglobulins constant domains may be selectedto promote symmetrical or asymmetrical complexes comprising at least onesingle-chain ALK4:ActRIIB trap.

In certain aspects, an ALK4:ActRIIB single-chain trap, or combinationsof such traps, may be used as ALK4:ActRIIB antagonists to treat orprevent an ALK4:ActRIIB disorder or disease as described herein.

It is understood that different elements of the fusion proteins (e.g.,immunoglobulin Fc fusion proteins) may be arranged in any manner that isconsistent with desired functionality. For example, an ALK4 and/orActRIIB polypeptide domain may be placed C-terminal to a heterologousdomain, or alternatively, a heterologous domain may be placed C-terminalto an ALK4 and/or ActRIIB polypeptide domain. The ALK4 and/or ActRIIBpolypeptide domain and the heterologous domain need not be adjacent in afusion protein, and additional domains or amino acid sequences may beincluded C- or N-terminal to either domain or between the domains.

For example, an ALK4 and/or ActRIIB receptor fusion protein may comprisean amino acid sequence as set forth in the formula A-B-C. The B portioncorresponds to an ALK4 or ActRIIB polypeptide domain. The A and Cportions may be independently zero, one, or more than one amino acid,and both the A and C portions when present are heterologous to B. The Aand/or C portions may be attached to the B portion via a linkersequence. A linker may be rich in glycine (e.g., 2-10, 2-5, 2-4, 2-3glycine residues) or glycine and proline residues and may, for example,contain a single sequence of threonine/serine and glycines or repeatingsequences of threonine/serine and/or glycines, e.g., GGG (SEQ ID NO:13), GGGG (SEQ ID NO: 14), TGGGG(SEQ ID NO: 15), SGGGG(SEQ ID NO: 16),TGGG(SEQ ID NO: 17), SGGG(SEQ ID NO: 18), or GGGGS (SEQ ID NO: 58)singlets, or repeats. In certain embodiments, an ALK4 and/or ActRIIBfusion protein comprises an amino acid sequence as set forth in theformula A-B-C, wherein A is a leader (signal) sequence, B consists of anALK4 and/or ActRIIB polypeptide domain, and C is a polypeptide portionthat enhances one or more of in vivo stability, in vivo half-life,uptake/administration, tissue localization or distribution, formation ofprotein complexes, and/or purification. In certain embodiments, an ALK4and/or ActRIIB fusion protein comprises an amino acid sequence as setforth in the formula A-B-C, wherein A is a TPA leader sequence, Bconsists of a ALK4 or ActRIIB receptor polypeptide domain, and C is animmunoglobulin Fc domain. Preferred fusion proteins comprise the aminoacid sequence set forth in any one of SEQ ID NOs: 39, 41, 42, 44, 45,46, 47, and 48.

In some embodiments, ALK4:ActRIIB heteromultimers further comprise oneor more heterologous portions (domains) so as to confer a desiredproperty. For example, some fusion domains are particularly useful forisolation of the fusion proteins by affinity chromatography. Well-knownexamples of such fusion domains include, but are not limited to,polyhistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin,protein A, protein G, an immunoglobulin heavy-chain constant region(Fc), maltose binding protein (MBP), or human serum albumin. For thepurpose of affinity purification, relevant matrices for affinitychromatography, such as glutathione-, amylase-, and nickel- orcobalt-conjugated resins are used. Many of such matrices are availablein “kit” form, such as the Pharmacia GST purification system and theQIAexpress™ system (Qiagen) useful with (HIS₆) fusion partners (“HIS₆”disclosed as SEQ ID NO: 96). As another example, a fusion domain may beselected so as to facilitate detection of the ligand trap polypeptides.Examples of such detection domains include the various fluorescentproteins (e.g., GFP) as well as “epitope tags,” which are usually shortpeptide sequences for which a specific antibody is available. Well-knownepitope tags for which specific monoclonal antibodies are readilyavailable include FLAG, influenza virus haemagglutinin (HA), and c-myctags. In some cases, the fusion domains have a protease cleavage site,such as for factor Xa or thrombin, which allows the relevant protease topartially digest the fusion proteins and thereby liberate therecombinant proteins therefrom. The liberated proteins can then beisolated from the fusion domain by subsequent chromatographicseparation.

In certain embodiments, ALK4 and/or ActRIIB polypeptides may contain oneor more modifications that are capable of stabilizing the polypeptides.For example, such modifications enhance the in vitro half-life of thepolypeptides, enhance circulatory half-life of the polypeptides, and/orreduce proteolytic degradation of the polypeptides. Such stabilizingmodifications include, but are not limited to, fusion proteins(including, for example, fusion proteins comprising an ALK4 and/orActRIIB polypeptide domain and a stabilizer domain), modifications of aglycosylation site (including, for example, addition of a glycosylationsite to a polypeptide of the disclosure), and modifications ofcarbohydrate moiety (including, for example, removal of carbohydratemoieties from a polypeptide of the disclosure). As used herein, the term“stabilizer domain” not only refers to a fusion domain (e.g., animmunoglobulin Fc domain) as in the case of fusion proteins, but alsoincludes nonproteinaceous modifications such as a carbohydrate moiety,or nonproteinaceous moiety, such as polyethylene glycol.

In preferred embodiments, ALK4:ActRIIB heteromultimers to be used inaccordance with the methods described herein are isolated complexes. Asused herein, an isolated protein (or protein complex) or polypeptide (orpolypeptide complex) is one which has been separated from a component ofits natural environment. In some embodiments, a heteromultimer of thedisclosure is purified to greater than 95%, 96%, 97%, 98%, or 99% purityas determined by, for example, electrophoretic (e.g., SDS-PAGE,isoelectric focusing (IEF), capillary electrophoresis) orchromatographic (e.g., ion exchange or reverse phase HPLC). Methods forassessment of antibody purity are well known in the art [Flatman et al.,(2007) J. Chromatogr. B 848:79-87]. In some embodiments, ALK4:ActRIIBheteromultimer preparations are substantially free of ALK4 and/orActRIIB homomultimers. For example, in some embodiments, ALK4:ActRIIBheteromultimer preparations comprise less than about 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or less than 1% ALK4 homomultimers. In someembodiments, ALK4:ActRIIB heteromultimer preparations comprise less thanabout 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% ActRIIBhomomultimers. In some embodiments, ALK4:ActRIIB heteromultimerpreparations comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, or less than 1% ALK4 homomultimers and less than about 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, or less than 1% ActRIIB homomultimers.

In certain embodiments, ALK4 and/or ActRIIB polypeptides, as well asheteromultimers comprising the same, of the disclosure can be producedby a variety of art-known techniques. For example, polypeptides can besynthesized using standard protein chemistry techniques such as thosedescribed in Bodansky, M. Principles of Peptide Synthesis, SpringerVerlag, Berlin (1993) and Grant G. A. (ed.), Synthetic Peptides: AUser's Guide, W. H. Freeman and Company, New York (1992). In addition,automated peptide synthesizers are commercially available (AdvancedChemTech Model 396; Milligen/Biosearch 9600). Alternatively, thepolypeptides, including fragments or variants thereof, may berecombinantly produced using various expression systems [E. coli,Chinese Hamster Ovary (CHO) cells, COS cells, baculovirus] as is wellknown in the art. In a further embodiment, the modified or unmodifiedpolypeptides may be produced by digestion of recombinantly producedfull-length ALK4 and/or ActRIIB polypeptides by using, for example, aprotease, e.g., trypsin, thermolysin, chymotrypsin, pepsin, or pairedbasic amino acid converting enzyme (PACE). Computer analysis (usingcommercially available software, e.g., MacVector, Omega, PCGene,Molecular Simulation, Inc.) can be used to identify proteolytic cleavagesites.

B. Nucleic Acids Encoding ALK4 and/or ActRIIB Polypeptides

In certain embodiments, the present disclosure provides isolated and/orrecombinant nucleic acids encoding ALK4 and/or ActRIIB receptors(including fragments, functional variants, and fusion proteins thereof)disclosed herein. For example, SEQ ID NO: 11 encodes a naturallyoccurring human ALK4 precursor polypeptide, while SEQ ID NO: 12 encodesthe mature extracellular domain of ALK4. The subject nucleic acids maybe single-stranded or double stranded. Such nucleic acids may be DNA orRNA molecules. These nucleic acids may be used, for example, in methodsfor making ALK4:ActRIIB heteromultimers as described herein.

As used herein, isolated nucleic acid(s) refers to a nucleic acidmolecule that has been separated from a component of its naturalenvironment. An isolated nucleic acid includes a nucleic acid moleculecontained in cells that ordinarily contain the nucleic acid molecule,but the nucleic acid molecule is present extrachromosomally or at achromosomal location that is different from its natural chromosomallocation.

In certain embodiments, nucleic acids encoding ALK4 and/or ActRIIBpolypeptides of the present disclosure are understood to include any oneof SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, or 43, as well as variantsthereof. Variant nucleotide sequences include sequences that differ byone or more nucleotide substitutions, additions, or deletions includingallelic variants, and therefore, will include coding sequences thatdiffer from the nucleotide sequence designated in any one of SEQ ID NOs:7, 8, 11, 12, 21, 22, 40, 43.

In certain embodiments, TGFβ superfamily ALK4 and/or ActRIIBpolypeptides of the present disclosure are encoded by isolated orrecombinant nucleic acid sequences that comprise, consist essentiallyof, or consists of a sequence that is least 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, or 43. One ofordinary skill in the art will appreciate that nucleic acid sequencesthat comprise, consist essentially of, or consists of a sequencecomplementary to a sequence that is least 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, or 43 also within thescope of the present disclosure. In further embodiments, the nucleicacid sequences of the disclosure can be isolated, recombinant, and/orfused with a heterologous nucleotide sequence or in a DNA library.

In other embodiments, nucleic acids of the present disclosure alsoinclude nucleotide sequences that hybridize under stringent conditionsto the nucleotide sequence designated in SEQ ID NOs: 7, 8, 11, 12, 21,22, 40, or 43, the complement sequence of SEQ ID NOs: 7, 8, 11, 12, 21,22, 40, or 43, or fragments thereof. One of ordinary skill in the artwill understand readily that appropriate stringency conditions whichpromote DNA hybridization can be varied. For example, one could performthe hybridization at 6.0× sodium chloride/sodium citrate (SSC) at about45° C., followed by a wash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In one embodiment, the disclosureprovides nucleic acids which hybridize under low stringency conditionsof 6×SSC at room temperature followed by a wash at 2×SSC at roomtemperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, or 43 to degeneracy in thegenetic code are also within the scope of the disclosure. For example, anumber of amino acids are designated by more than one triplet. Codonsthat specify the same amino acid, or synonyms (for example, CAU and CACare synonyms for histidine) may result in “silent” mutations which donot affect the amino acid sequence of the protein. However, it isexpected that DNA sequence polymorphisms that do lead to changes in theamino acid sequences of the subject proteins will exist among mammaliancells. One skilled in the art will appreciate that these variations inone or more nucleotides (up to about 3-5% of the nucleotides) of thenucleic acids encoding a particular protein may exist among individualsof a given species due to natural allelic variation. Any and all suchnucleotide variations and resulting amino acid polymorphisms are withinthe scope of this disclosure.

In certain embodiments, the recombinant nucleic acids of the disclosuremay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the disclosure. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In some embodiments, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selectable marker genes are well known in the art and will vary with thehost cell used.

In certain aspects, the subject nucleic acid is provided in anexpression vector comprising a nucleotide sequence encoding an ALK4and/or ActRIIB polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of ALK4 and/or ActRIIB polypeptide.Accordingly, the term regulatory sequence includes promoters, enhancers,and other expression control elements. Exemplary regulatory sequencesare described in Goeddel; Gene Expression Technology: Methods inEnzymology, Academic Press, San Diego, Calif. (1990). For instance, anyof a wide variety of expression control sequences that control theexpression of a DNA sequence when operatively linked to it may be usedin these vectors to express DNA sequences encoding a ALK4 and/or ActRIIBpolypeptides. Such useful expression control sequences, include, forexample, the early and late promoters of SV40, tet promoter, adenovirusor cytomegalovirus immediate early promoter, RSV promoters, the lacsystem, the trp system, the TAC or TRC system, T7 promoter whoseexpression is directed by T7 RNA polymerase, the major operator andpromoter regions of phage lambda, the control regions for fd coatprotein, the promoter for 3-phosphoglycerate kinase or other glycolyticenzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters ofthe yeast α-mating factors, the polyhedron promoter of the baculovirussystem and other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof. It should be understood that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transformed and/or the type of protein desired to beexpressed. Moreover, the vector's copy number, the ability to controlthat copy number and the expression of any other protein encoded by thevector, such as antibiotic markers, should also be considered.

A recombinant nucleic acid of the present disclosure can be produced byligating the cloned gene, or a portion thereof, into a vector suitablefor expression in either prokaryotic cells, eukaryotic cells (yeast,avian, insect or mammalian), or both. Expression vehicles for productionof a recombinant ALK4 and/or ActRIIB polypeptides include plasmids andother vectors. For instance, suitable vectors include plasmids of thefollowing types: pBR322-derived plasmids, pEMBL-derived plasmids,pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmidsfor expression in prokaryotic cells, such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures [Molecular Cloning A Laboratory Manual, 3rd Ed.,ed. by Sambrook, Fritsch and Maniatis Cold Spring Harbor LaboratoryPress, 2001]. In some instances, it may be desirable to express therecombinant polypeptides by the use of a baculovirus expression system.Examples of such baculovirus expression systems include pVL-derivedvectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors(such as pAcUW1), and pBlueBac-derived vectors (such as the β-galcontaining pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject ALK4 and/or ActRIIB polypeptides in CHO cells, such as aPcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors(Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison,Wis.). As will be apparent, the subject gene constructs can be used tocause expression of the subject ALK4 and/or ActRIIB polypeptide in cellspropagated in culture, e.g., to produce proteins, including fusionproteins or variant proteins, for purification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence for one or more of thesubject ALK4 and/or ActRIIB polypeptides. The host cell may be anyprokaryotic or eukaryotic cell. For example, an ALK4 and/or ActRIIBpolypeptide may be expressed in bacterial cells such as E. coli, insectcells (e.g., using a baculovirus expression system), yeast, or mammaliancells [e.g. a Chinese hamster ovary (CHO) cell line]. Other suitablehost cells are known to those skilled in the art.

Accordingly, the present disclosure further pertains to methods ofproducing the subject ALK4 and/or ActRIIB polypeptides. For example, ahost cell transfected with an expression vector encoding an ALK4 and/orActRIIB polypeptide can be cultured under appropriate conditions toallow expression of the ALK4 and/or ActRIIB polypeptide to occur. Thepolypeptide may be secreted and isolated from a mixture of cells andmedium containing the polypeptide. Alternatively, ALK4 and/or ActRIIBpolypeptide may be isolated from a cytoplasmic or membrane fractionobtained from harvested and lysed cells. A cell culture includes hostcells, media and other byproducts. Suitable media for cell culture arewell known in the art. The subject polypeptides can be isolated fromcell culture medium, host cells, or both, using techniques known in theart for purifying proteins, including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis,immunoaffinity purification with antibodies specific for particularepitopes of ALK4 and/or ActRIIB polypeptides and affinity purificationwith an agent that binds to a domain fused to ALK4 and/or ActRIIBpolypeptide (e.g., a protein A column may be used to purify ALK4-Fcand/or ActRIIB-Fc fusion proteins). In some embodiments, the ALK4 and/orActRIIB polypeptide is a fusion protein containing a domain whichfacilitates its purification.

In some embodiments, purification is achieved by a series of columnchromatography steps, including, for example, three or more of thefollowing, in any order: protein A chromatography, Q sepharosechromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange. An ALK4and/or ActRIIB polypeptides, as well as fusion proteins thereof, may bepurified to a purity of >90%, >95%, >96%, >98%, or >99% as determined bysize exclusion chromatography and >90%, >95%, >96%, >98%, or >99% asdetermined by SDS PAGE. The target level of purity should be one that issufficient to achieve desirable results in mammalian systems,particularly non-human primates, rodents (mice), and humans.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant ALK4 and/orActRIIB polypeptide, can allow purification of the expressed fusionprotein by affinity chromatography using a Ni²⁺ metal resin. Thepurification leader sequence can then be subsequently removed bytreatment with enterokinase to provide the purified ALK4 and/or ActRIIBpolypeptide, as well as heteromultimers thereof [Hochuli et al. (1987)J. Chromatography 411:177; and Janknecht et al. (1991) PNAS USA88:8972].

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence. See,e.g., Current Protocols in Molecular Biology, eds. Ausubel et al., JohnWiley & Sons: 1992.

C. Antibody Antagonists

In certain aspects, an ALK4:ActRIIB antagonist is an antibody(ALK4:ActRIIB antagonist antibody), or combination of antibodies. AnALK4:ActRIIB antagonist antibody, or combination of antibodies, may bindto, for example, one or more ALK4 ligands, ActRIIB ligands,ALK4:ActRIIB-binding ligands, an ALK4 receptor, an ActRIIB receptor,and/or one or more TGF-beta superfamily co-receptors. As describedherein, ALK4:ActRIIB antagonist antibodies may be used alone, or incombination with one or more supportive therapies or active agents, totreat a in need thereof (e.g., a subject with a bone-related disease orcondition, a muscle related disease or condition, or a disease orcondition associated with excess or unwanted fat)

In certain aspects, an ALK4:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits one or more of the ligandsbound or likely bound by an ALK4:ActRIIB heteromultimer, such as activinB, GDF11, activin A, GDF8, BMP10, BMP6, and GDF3. Therefore, in someembodiments, an ALK4:ActRIIB antagonist antibody, or combination ofantibodies, binds to at least one of such ligands. As an example, asused herein, an activin B antibody (or anti-activin B antibody)generally refers to an antibody that can bind to activin B withsufficient affinity such that the antibody is useful as a diagnosticand/or therapeutic agent in targeting activin B. In certain embodiments,the extent of binding of a activin B antibody to an unrelated,non-activin B protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%,3%, 2%, or less than about 1% of the binding of the antibody to activinB as measured, for example, by a radioimmunoassay (RIA), Biacore, orother protein interaction or binding affinity assay. In certainembodiments, an activin B antibody binds to an epitope of activin B thatis conserved among activin B from different species. In certainpreferred embodiments, an anti-activin B antibody binds to human activinB. In some embodiments, an activin B antibody may inhibit activin B frombinding to a type I and/or type II receptor (e.g., ActRIIB and/or ALK4)and thus inhibit activin B-mediated signaling (e.g., Smad signaling). Insome embodiments, an activin B antibody may inhibit activin B frombinding to a co-receptor and thus inhibit activin B-mediated signaling(e.g., Smad signaling). It should be noted that activin B shares somesequence homology to activin A, C and E and therefore antibodies thatbind to activin B, in some instances, may also bind to and/or inhibitanother activin. In some embodiments, the disclosure relates to amultispecific antibody (e.g., bi-specific antibody), and uses thereof,that binds to, for example, activin B and further binds to, for example,one or more additional TGF-β superfamily ligands that bind toALK4:ActRIIB heteromultimer [e.g., activin (e.g., activin A, activin AB,and activin B), GDF11, GDF8, BMP10, BMP6, and GDF3], one or more type Ireceptor and/or type II receptors (e.g., ActRIIB and/or ALK4), and/orone or more co-receptors. In some embodiments, a multispecific antibodythat binds to activin B does not bind or does not substantially bind toBMP9 (e.g., binds to BMP9 with a K_(D) of greater than 1×10⁻⁷ M or hasrelatively modest binding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). Insome embodiments, the disclosure relates to combinations of antibodies,and uses thereof, wherein the combination of antibodies comprises acombination of antibodies that bind to, for example, two or more TGF-βsuperfamily ligand that bind to ALK4:ActRIIB heteromultimer [e.g.,activin (e.g., activin A, activin B, and activin AB), GDF11, GDF8,BMP10, BMP6, and GDF] one or more type I receptor and/or type IIreceptors (e.g., ActRIIB and/or ALK4), and/or one or more co-receptors.In some embodiments, a combination of antibodies does not comprise aBMP9 antibody.

In certain aspects, an ALK4:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least GDF8. Therefore, insome embodiments, an ALK4:ActRIIB antagonist antibody, or combination ofantibodies, binds to at least GDF8. As used herein, a GDF8 antibody (oranti-GDF8 antibody) generally refers to an antibody that binds to GDF8with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting GDF8. In certainembodiments, the extent of binding of a GDF8 antibody to an unrelated,non-GDF8 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,or less than about 1% of the binding of the antibody to GDF8 asmeasured, for example, by a radioimmunoassay (RIA), Biacore, or otherprotein interaction or binding affinity assay. In certain embodiments, aGDF8 antibody binds to an epitope of GDF8 that is conserved among GDF8from different species. In certain preferred embodiments, an anti-GDF8antibody binds to human GDF8. In some embodiments, a GDF8 antibody mayinhibit GDF8 from binding to a type I and/or type II receptor (e.g.,ActRIIB and/or ALK4) and thus inhibit GDF8-mediated signaling (e.g.,Smad signaling). In some embodiments, a GDF8 antibody may inhibit GDF8from binding to a co-receptor and thus inhibit GDF8-mediated signaling(e.g., Smad signaling). It should be noted that GDF8 has high sequencehomology to GDF11 and therefore antibodies that bind to GDF8, in someinstances, may also bind to and/or inhibit GDF11. In some embodiments,the disclosure relates to a multispecific antibody (e.g., bi-specificantibody), and uses thereof, that binds to GDF8 and further binds to,for example, one or more additional TGF-β superfamily ligands that bindto ALK4:ActRIIB heteromultimer [e.g., activin (e.g., activin A, activinB, and activin AB), GDF11, BMP10, BMP6, and GDF3], one or more type Ireceptor and/or type II receptors (e.g., ActRIIB and/or ALK4), and/orone or more co-receptors. In some embodiments, a multispecific antibodythat binds to GDF8 does not bind or does not substantially bind to BMP9(e.g., binds to BMP9 with a K_(D) of greater than 1×10⁻⁷M or hasrelatively modest binding, e.g., about 1×10⁻⁸M or about 1×10⁻⁹M). Insome embodiments, the disclosure relates to combinations of antibodies,and uses thereof, wherein the combination of antibodies comprises a GDF8antibody and one or more additional antibodies that bind to, forexample, one or more additional TGF-β superfamily ligands that bind toALK4:ActRIIB heteromultimer [e.g., activin (e.g., activin A, activin B,and activin AB), GDF11, GDF3, BMP6, and BMP10], one or more type Ireceptor and/or type II receptors (e.g., ActRIIB and/or ALK4), and/orone or more co-receptors. In some embodiments, a combination ofantibodies that comprises a GDF8 antibody does not comprise a BMP9antibody.

In certain aspects, an ALK4:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least GDF11. Therefore,in some embodiments, an ALK4:ActRIIB antagonist antibody, or combinationof antibodies, binds to at least GDF11. As used herein, a GDF11 antibody(or anti-GDF11 antibody) generally refers to an antibody that binds toGDF11 with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting GDF11. In certainembodiments, the extent of binding of a GDF11 antibody to an unrelated,non-GDF11 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, or less than about 1% of the binding of the antibody to GDF11 asmeasured, for example, by a radioimmunoassay (RIA), Biacore, or otherprotein interaction or binding affinity assay. In certain embodiments, aGDF11 antibody binds to an epitope of GDF11 that is conserved amongGDF11 from different species. In certain preferred embodiments, ananti-GDF11 antibody binds to human GDF11. In some embodiments, a GDF11antibody may inhibit GDF11 from binding to a type I and/or type IIreceptor (e.g., ActRIIB and/or ALK4) and thus inhibit GDF11-mediatedsignaling (e.g., Smad signaling). In some embodiments, a GDF11 antibodymay inhibit GDF11 from binding to a co-receptor and thus inhibitGDF11-mediated signaling (e.g., Smad signaling). It should be noted thatGDF11 has high sequence homology to GDF8 and therefore antibodies thatbind to GDF11, in some instances, may also bind to and/or inhibit GDF8.In some embodiments, the disclosure relates to a multispecific antibody(e.g., bi-specific antibody), and uses thereof, that binds to GDF11 andfurther binds to, for example, one or more additional TGF-β superfamilyligands that bind to ALK4:ActRIIB heteromultimer [e.g., activin (e.g.,activin A, activin B, and activin AB), GDF8, BMP10, BMP6, and GDF3], oneor more type I receptor and/or type II receptors (e.g., ActRIIB and/orALK4), and/or one or more co-receptors. In some embodiments, amultispecific antibody that binds to GDF11 does not bind or does notsubstantially bind to BMP9 (e.g., binds to BMP9 with a K_(D) of greaterthan 1×10⁻⁷M or has relatively modest binding, e.g., about 1×10⁻⁸M orabout 1×10⁻⁹M). In some embodiments, the disclosure relates tocombinations of antibodies, and uses thereof, wherein the combination ofantibodies comprises a GDF11 antibody and one or more additionalantibodies that bind to, for example, one or more additional TGF-βsuperfamily ligands that bind to ALK4:ActRIIB heteromultimer [e.g.,activin (e.g., activin A, activin B, and activin AB), GDF8, GDF3, BMP6,and BMP10], one or more type I receptor and/or type II receptors (e.g.,ActRIIB and/or ALK4), and/or one or more co-receptors. In someembodiments, a combination of antibodies that comprises a GDF11 antibodydoes not comprise a BMP9 antibody.

In certain aspects, an ALK4:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least GDF3. Therefore, insome embodiments, an ALK4:ActRIIB antagonist antibody, or combination ofantibodies, binds to at least GDF3. As used herein, a GDF3 antibody (oranti-GDF3 antibody) generally refers to an antibody that binds to GDF3with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting GDF3. In certainembodiments, the extent of binding of a GDF3 antibody to an unrelated,non-GDF3 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,or less than about 1% of the binding of the antibody to GDF3 asmeasured, for example, by a radioimmunoassay (RIA), Biacore, or otherprotein interaction or binding affinity assay. In certain embodiments, aGDF3 antibody binds to an epitope of GDF3 that is conserved among GDF3from different species. In certain preferred embodiments, an anti-GDF3antibody binds to human GDF3. In some embodiments, a GDF3 antibody mayinhibit GDF3 from binding to a type I and/or type II receptor (e.g.,ActRIIB and/or ALK4) and thus inhibit GDF3-mediated signaling (e.g.,Smad signaling). In some embodiments, a GDF3 antibody may inhibit GDF3from binding to a co-receptor and thus inhibit GDF3-mediated signaling(e.g., Smad signaling). In some embodiments, the disclosure relates to amultispecific antibody (e.g., bi-specific antibody), and uses thereof,that binds to GDF3 and further binds to, for example, one or moreadditional TGF-β superfamily ligands that bind to ALK4:ActRIIBheteromultimer [e.g., activin (e.g., activin A, activin B, and activinAB), GDF8, BMP10, BMP6, and GDF11], one or more type I receptor and/ortype II receptors (e.g., ActRIIB and/or ALK4), and/or one or moreco-receptors. In some embodiments, a multispecific antibody that bindsto GDF3 does not bind or does not substantially bind to BMP9 (e.g.,binds to BMP9 with a K_(D) of greater than 1×10⁻⁷M or has relativelymodest binding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). In someembodiments, the disclosure relates to combinations of antibodies, anduses thereof, wherein the combination of antibodies comprises a GDF3antibody and one or more additional antibodies that bind to, forexample, one or more additional TGF-β superfamily ligands that bind toALK4:ActRIIB heteromultimer [e.g., activin (e.g., activin A, activin B,and activin AB), GDF8, GDF11, BMP6, and BMP10], one or more type Ireceptor and/or type II receptors (e.g., ActRIIB and/or ALK4), and/orone or more co-receptors. In some embodiments, a combination ofantibodies that comprises a GDF3 antibody does not comprise a BMP9antibody.

In certain aspects, an ALK4:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least BMP6. Therefore, insome embodiments, an ALK4:ActRIIB antagonist antibody, or combination ofantibodies, binds to at least BMP6. As used herein, a BMP6 antibody (oranti-BMP6 antibody) generally refers to an antibody that binds to BMP6with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting BMP6. In certainembodiments, the extent of binding of a BMP6 antibody to an unrelated,non-BMP6 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,or less than about 1% of the binding of the antibody to BMP6 asmeasured, for example, by a radioimmunoassay (RIA), Biacore, or otherprotein interaction or binding affinity assay. In certain embodiments, aBMP6 antibody binds to an epitope of BMP6 that is conserved among BMP6from different species. In certain preferred embodiments, an anti-BMP6antibody binds to human BMP6. In some embodiments, a BMP6 antibody mayinhibit BMP6 from binding to a type I and/or type II receptor (e.g.,ActRIIB and/or ALK4) and thus inhibit BMP6-mediated signaling (e.g.,Smad signaling). In some embodiments, a BMP6 antibody may inhibit BMP6from binding to a co-receptor and thus inhibit BMP6-mediated signaling(e.g., Smad signaling). In some embodiments, the disclosure relates to amultispecific antibody (e.g., bi-specific antibody), and uses thereof,that binds to BMP6 and further binds to, for example, one or moreadditional TGF-β superfamily ligands that bind to ALK4:ActRIIBheteromultimer [e.g., activin (e.g., activin A, activin B, and activinAB), GDF8, BMP10, GDF3, and GDF11], one or more type I receptor and/ortype II receptors (e.g., ActRIIB and/or ALK4), and/or one or moreco-receptors. In some embodiments, a multispecific antibody that bindsto BMP6 does not bind or does not substantially bind to BMP9 (e.g.,binds to BMP9 with a K_(D) of greater than 1×10⁻⁷M or has relativelymodest binding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). In someembodiments, the disclosure relates to combinations of antibodies, anduses thereof, wherein the combination of antibodies comprises a BMP6antibody and one or more additional antibodies that bind to, forexample, one or more additional TGF-β superfamily ligands that bind toALK4:ActRIIB heteromultimer [e.g., activin (e.g., activin A, activin B,and activin AB), GDF8, GDF11, GDF3, and BMP10], one or more type Ireceptor and/or type II receptors (e.g., ActRIIB and/or ALK4), and/orone or more co-receptors. In some embodiments, a combination ofantibodies that comprises a BMP6 antibody does not comprise a BMP9antibody.

In certain aspects, an ALK4:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least BMP10. Therefore,in some embodiments, an ALK4:ActRIIB antagonist antibody, or combinationof antibodies, binds to at least BMP10. As used herein, a BMP10 antibody(or anti-BMP10 antibody) generally refers to an antibody that binds toBMP10 with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting BMP10. In certainembodiments, the extent of binding of a BMP10 antibody to an unrelated,non-BMP10 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, or less than about 1% of the binding of the antibody to BMP10 asmeasured, for example, by a radioimmunoassay (RIA), Biacore, or otherprotein interaction or binding affinity assay. In certain embodiments, aBMP10 antibody binds to an epitope of BMP10 that is conserved amongBMP10 from different species. In certain preferred embodiments, ananti-BMP10 antibody binds to human BMP10. In some embodiments, a BMP10antibody may inhibit BMP10 from binding to a type I and/or type IIreceptor (e.g., ActRIIB and/or ALK4) and thus inhibit BMP10-mediatedsignaling (e.g., Smad signaling). In some embodiments, a BMP10 antibodymay inhibit BMP10 from binding to a co-receptor and thus inhibitBMP10-mediated signaling (e.g., Smad signaling). In some embodiments,the disclosure relates to a multispecific antibody (e.g., bi-specificantibody), and uses thereof, that binds to BMP10 and further binds to,for example, one or more additional TGF-β superfamily ligands that bindto ALK4:ActRIIB heteromultimer [e.g., activin (e.g., activin A, activinB, and activin AB), GDF8, BMP6, GDF3, and GDF11], one or more type Ireceptor and/or type II receptors (e.g., ActRIIB and/or ALK4), and/orone or more co-receptors. In some embodiments, a multispecific antibodythat binds to BMP10 does not bind or does not substantially bind to BMP9(e.g., binds to BMP9 with a K_(D) of greater than 1×10⁻⁷ M or hasrelatively modest binding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). Insome embodiments, the disclosure relates to combinations of antibodies,and uses thereof, wherein the combination of antibodies comprises aBMP10 antibody and one or more additional antibodies that bind to, forexample, one or more additional TGF-β superfamily ligands that bind toALK4:ActRIIB heteromultimer [e.g., activin (e.g., activin A, activin B,and activin AB), GDF8, GDF11, GDF3, and BMP6], one or more type Ireceptor and/or type II receptors (e.g., ActRIIB and/or ALK4), and/orone or more co-receptors. In some embodiments, a combination ofantibodies that comprises a BMP10 antibody does not comprise a BMP9antibody.

In certain aspects, an ALK4:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least activin (activin A,activin B, activin C, activin E, activin AB, activin AC, activin AE,activin BC and/or activin BE). Therefore, in some embodiments, anALK4:ActRIIB antagonist antibody, or combination of antibodies, binds toat least activin (activin A, activin B, activin C, activin E, activinAB, activin AC, activin AE, activin BC and/or activin BE). As usedherein, an activin antibody (or anti-activin antibody) generally refersto an antibody that can bind to a form of activin with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting that form of activin. In certainembodiments, the extent of binding of an activin antibody to anunrelated, non-activin protein is less than about 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody toactivin as measured, for example, by a radioimmunoassay (RIA), Biacore,or other protein interaction or binding affinity assay. In certainembodiments, an activin antibody binds to an epitope of activin that isconserved among activin from different species. In certain preferredembodiments, an anti-activin antibody binds to human activin. In otherpreferred embodiments, a activin antibody may inhibit activin frombinding to a type I and/or type II receptor (e.g., ActRIIB and/or ALK4)and thus inhibit activin-mediated signaling (e.g., Smad signaling). Insome embodiments, an activin antibody binds to activin B. In someembodiments, an activin antibody binds to activin A. In someembodiments, an activin antibody binds to activin A and activin B. Insome embodiments, an activin antibody binds to activin AB. In someembodiments, an activin antibody binds to activin C. In someembodiments, an activin antibody binds to activin E. In someembodiments, an activin antibody binds to activin A and activin C. Insome embodiments, an activin antibody binds to activin AC. In someembodiments, an activin antibody binds to activin A and activin E. Insome embodiments, an activin antibody binds to activin AE. In someembodiments, an activin antibody binds to activin B and activin C. Insome embodiments, an activin antibody binds to activin BC. In someembodiments, an activin antibody binds to activin B and activin E. Insome embodiments, an activin antibody binds to activin BE. In someembodiments, an activin antibody binds to activin A, activin B, andactivin C. In some embodiments, an activin antibody binds to activin A,activin B, and activin E. Optionally, an activin antibody that binds toone or more of activin A, activin B, and activin C may further bind toactivin E. In some embodiments, the disclosure relates to amultispecific antibody (e.g., bi-specific antibody), and uses thereof,that binds to activin and further binds to, for example, one or moreadditional TGF-β superfamily ligands that bind to an ALK4:ActRIIBheteromultimer [e.g., GDF11, GDF8, BMP10, BMP6, and GDF3], one or moretype I receptor and/or type II receptors (e.g., ActRIIB and/or ALK4),and/or one or more co-receptors. In some embodiments, a multispecificantibody that binds to activin does not bind or does not substantiallybind to BMP9 (e.g., binds to BMP9 with a K_(D) of greater than 1×10⁻⁷ Mor has relatively modest binding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹M. In some embodiments, the disclosure relates to combinations ofantibodies, and uses thereof, wherein the combination of antibodiescomprises an activin antibody and one or more additional antibodies thatbind to, for example, one or more additional TGF-β superfamily ligandthat bind to an ALK4:ActRIIB heteromultimer [e.g., GDF11, GDF8 GDF3,BMP6, and BMP10], one or more type I receptor and/or type II receptors(e.g., ActRIIB and/or ALK4), and/or one or more co-receptors. In someembodiments, a combination of antibodies that comprises an activinantibody does not comprise a BMP9 antibody.

With respect to antibodies that bind to and antagonize ligands that bindto ALK4:ActRIIB, [e.g., activin (e.g., activin A, activin B, and activinAB), GDF8, GDF3, BMP6, GDF11, and BMP10], it is contemplated that anantibody may be designed as a bispecific antibody comprising a firstportion that binds to an epitope of such ligand, such that the firstportion of the antibody competes for binding with a type I receptor andcomprising a second portion that binds to an epitope of such ligand,such that the second portion of the antibody competes for binding with atype II receptor. In this manner, a bispecific antibody targeting asingle ligand can be designed to mimic the dual type I-type II receptorbinding blockade that may be conferred by an ALK4:ActRIIBheteromultimer. Similarly it is contemplated that the same effect couldbe achieved using a combination of two or more antibodies wherein atleast a first antibody binds to an epitope of such ligand, such that thefirst antibody competes for binding with a type I receptor and at leasta second antibody binds to an epitope of such ligand, such that thesecond antibody competes for binding with a type II receptor.

In certain aspects, an ALK4:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least ActRIIB Therefore,in some embodiments, an ALK4:ActRIIB antagonist antibody, or combinationof antibodies, binds to at least ActRIIB As used herein, an ActRIIBantibody (anti-ActRIIB antibody) generally refers to an antibody thatbinds to ActRIIB with sufficient affinity such that the antibody isuseful as a diagnostic and/or therapeutic agent in targeting ActRIIB Incertain embodiments, the extent of binding of an anti-ActRIIB antibodyto an unrelated, non-ActRIIB protein is less than about 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibodyto ActRIIB as measured, for example, by a radioimmunoassay (RIA),Biacore, or other protein-protein interaction or binding affinity assay.In certain embodiments, an anti-ActRIIB antibody binds to an epitope ofActRIIB that is conserved among ActRIIB from different species. Incertain preferred embodiments, an anti-ActRIIB antibody binds to humanActRIIB In some embodiments, an anti-ActRIIB antibody may inhibit one ormore TGF-β superfamily ligands that bind to ALK4:ActRIIB heteromultimers[e.g., GDF8, activin (e.g., activin A, activin B, and activin AB) GDF3,BMP6, and BMP10] from binding to ActRIIB and/or ALK4. In someembodiments, an anti-ActRIIB antibody is a multispecific antibody (e.g.,bi-specific antibody) that binds to ActRIIB and one or more TGF-βsuperfamily ligands that bind to ALK4:ActRIIB heteromultimers [e.g.,GDF11, GDF8, activin (e.g., activin A, activin B, and, activin AB) GDF3,BMP6, and BMP10], type I receptor (e.g., ALK4), co-receptor, and/or anadditional type II receptor. In some embodiments, the disclosure relatesto combinations of antibodies, and uses thereof, wherein the combinationof antibodies comprises an anti-ActRIIB antibody and one or moreadditional antibodies that bind to, for example, one or more TGF-βsuperfamily ligands that bind to ALK4:ActRIIB heteromultimers [e.g.,GDF11, GDF8, activin (e.g., activin A, activin B, and activin AB) GDF3,BMP6, BMP10, nodal, and BMP9], co-receptors, type I receptors (e.g.,ALK4), and/or additional type II receptors. It should be noted thatActRIIB has sequence similarity to ActRIIA and therefore antibodies thatbind to ActRIIB, in some instances, may also bind to and/or inhibitActRIIA.

In certain aspects, an ALK4:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least ALK4. Therefore, insome embodiments, an ALK4:ActRIIB antagonist antibody, or combination ofantibodies, binds to at least ALK4. As used herein, an ALK4 antibody(anti-ALK4 antibody) generally refers to an antibody that binds to ALK4with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting ALK4. In certainembodiments, the extent of binding of an anti-ALK4 antibody to anunrelated, non-ALK4 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, or less than about 1% of the binding of the antibody to ALK4as measured, for example, by a radioimmunoassay (RIA), Biacore, or otherprotein-protein interaction or binding affinity assay. In certainembodiments, an anti-ALK4 antibody binds to an epitope of ALK4 that isconserved among ALK4 from different species. In certain preferredembodiments, an anti-ALK4 antibody binds to human ALK4. In someembodiments, an anti-ALK4 antibody may inhibit one or more TGF-βsuperfamily ligands that bind to ALK4:ActRIIB heteromultimers [e.g.,GDF11, GDF8, activin (e.g., activin A, activin B, and activin AB) GDF3,BMP6, and BMP10] from binding to a type I receptor (e.g., ALK4), type IIreceptor (e.g., ActRIIB), or co-receptor. In some embodiments, ananti-ALK4 antibody is a multispecific antibody (e.g., bi-specificantibody) that binds to ALK4 and one or more TGF-β superfamily ligandsthat bind to ALK4:ActRIIB heteromultimers [e.g., activin (e.g., activinA, activin B, and activin AB), GDF11, GDF8, BMP10, BMP6, and GDF3], typeII receptors (e.g., ActRIIB), co-receptors, and/or an additional type Ireceptor. In some embodiments, the disclosure relates to combinations ofantibodies, and uses thereof, wherein the combination of antibodiescomprises an anti-ALK4 antibody and one or more additional antibodiesthat bind to, for example, one or more TGF-β superfamily ligands thatbind to ALK4:ActRIIB heteromultimers [e.g., activin (e.g., activin A,activin B, and activin AB), GDF11, GDF8, BMP10, BMP6, and GDF3],co-receptors, an additional type I receptor, and/or type II receptors(e.g., ActRIIB).

As described herein, there are a variety of methods for generatingheteromultimers. Such methods may be used to generate heteromultimerscomprising an antibody-binding domain (e.g., a complex of V_(L) andV_(H) chains) and one or more polypeptides selected from an ALK4polypeptide, an ActRIIB polypeptide, an ALK4:ActRIIB heteromer, or anALK4:ActRIIB single trap polypeptide. See FIGS. 10 and 12D. For example,in some embodiments, the present disclosure provides protein complexescomprising a ligand-binding domain of an antibody that binds to anALK4:ActRIIB-binding ligand [e.g., activin (e.g., activin A, activin B,and activin AB), GDF11, GDF8, BMP10, BMP6, and GDF3] which is covalentlyor non-covalently associated with an ALK4 polypeptide. In someembodiments, the disclosure provides protein complexes comprising aligand-binding domain of an antibody that binds to anALK4:ActRIIB-binding ligand [e.g., activin (e.g., activin A, activin B,and activin AB), GDF11, GDF8, BMP10, BMP6, and GDF3] which is covalentlyor non-covalently associated with an ActRIIB polypeptide. In someembodiments, the present disclosure provides protein complexescomprising a ligand-binding domain of an antibody that binds to anALK4:ActRIIB-binding ligand [e.g., activin (e.g., activin A, activin B,and activin AB), GDF11, GDF8, BMP10, BMP6, and GDF3] which is covalentlyor non-covalently associated with a ALK4:ActRIIB single-chain ligandtrap. In some embodiments, the present disclosure provides proteincomplexes comprising a ligand-binding domain of an antibody that bindsto an ALK4:ActRIIB-binding ligand covalently or non-covalentlyassociated with a ALK4:ActRIIB heteromultimer.

The term antibody is used herein in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity. An antibody fragment refers to amolecule other than an intact antibody that comprises a portion of anintact antibody that binds the antigen to which the intact antibodybinds. Examples of antibody fragments include, but are not limited to,Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies; linear antibodies;single-chain antibody molecules (e.g., scFv); and multispecificantibodies formed from antibody fragments [see, e.g., Hudson et al.(2003) Nat. Med. 9:129-134; Plückthun, in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, NewYork), pp. 269-315 (1994); WO 93/16185; and U.S. Pat. Nos. 5,571,894;5,587,458; and 5,869,046]. Diabodies are antibody fragments with twoantigen-binding sites that may be bivalent or bispecific [see, e.g., EP404,097; WO 1993/01161; Hudson et al. (2003) Nat. Med. 9:129-134 (2003);and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448].Triabodies and tetrabodies are also described in Hudson et al. (2003)Nat. Med. 9:129-134. Single-domain antibodies are antibody fragmentscomprising all or a portion of the heavy-chain variable domain or all ora portion of the light-chain variable domain of an antibody. In certainembodiments, a single-domain antibody is a human single-domain antibody[see, e.g., U.S. Pat. No. 6,248,516]. Antibodies disclosed herein may bepolyclonal antibodies or monoclonal antibodies. In certain embodiments,the antibodies of the present disclosure comprise a label attachedthereto and able to be detected (e.g., the label can be a radioisotope,fluorescent compound, enzyme, or enzyme co-factor). In certain preferredembodiments, the antibodies of the present disclosure are isolatedantibodies. In certain preferred embodiments, the antibodies of thepresent disclosure are recombinant antibodies.

The antibodies herein may be of any class. The class of an antibodyrefers to the type of constant domain or constant region possessed byits heavy chain. There are five major classes of antibodies: IgA, IgD,IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), for example, IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, andIgA₂. The heavy chain constant domains that correspond to the differentclasses of immunoglobulins are called alpha, delta, epsilon, gamma, andmu.

In general, an antibody for use in the methods disclosed hereinspecifically binds to its target antigen, preferably with high bindingaffinity. Affinity may be expressed as a K_(D) value and reflects theintrinsic binding affinity (e.g., with minimized avidity effects).Typically, binding affinity is measured in vitro, whether in a cell-freeor cell-associated setting. Any of a number of assays known in the art,including those disclosed herein, can be used to obtain binding affinitymeasurements including, for example, Biacore, radiolabeledantigen-binding assay (RIA), and ELISA. In some embodiments, antibodiesof the present disclosure bind to their target antigens (e.g. ALK4,ActRIIB, activin A, activin B, activin AB, GDF11, GDF8, BMP10, BMP6, andGDF3) with at least a K_(D) of 1×10⁻⁷ or stronger, 1×10⁻⁸ or stronger,1×10⁻⁹ or stronger, 1×10⁻¹⁰ or stronger, 1×10⁻¹¹ or stronger, 1×10⁻¹² orstronger, 1×10⁻¹³ or stronger, or 1×10⁻¹⁴ or stronger.

In certain embodiments, K_(D) is measured by RIA performed with the Fabversion of an antibody of interest and its target antigen as describedby the following assay. Solution binding affinity of Fabs for theantigen is measured by equilibrating Fab with a minimal concentration ofradiolabeled antigen (e.g., ¹²⁵I-labeled) in the presence of a titrationseries of unlabeled antigen, then capturing bound antigen with ananti-Fab antibody-coated plate [see, e.g., Chen et al. (1999) J. Mol.Biol. 293:865-881]. To establish conditions for the assay, multi-wellplates (e.g., MICROTITER® from Thermo Scientific) are coated (e.g.,overnight) with a capturing anti-Fab antibody (e.g., from Cappel Labs)and subsequently blocked with bovine serum albumin, preferably at roomtemperature (approximately 23° C.). In a non-adsorbent plate,radiolabeled antigen are mixed with serial dilutions of a Fab ofinterest [e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599]. The Fab ofinterest is then incubated, preferably overnight but the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation, preferably at room temperature for aboutone hour. The solution is then removed and the plate is washed timesseveral times, preferably with polysorbate 20 and PBS mixture. When theplates have dried, scintillant (e.g., MICROSCINT® from Packard) isadded, and the plates are counted on a gamma counter (e.g., TOPCOUNT®from Packard).

According to another embodiment, K_(D) is measured using surface plasmonresonance assays using, for example a BIACORE® 2000 or a BIACORE® 3000(BIAcore, Inc., Piscataway, N.J.) with immobilized antigen CM5 chips atabout 10 response units (RU). Briefly, carboxymethylated dextranbiosensor chips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions. Forexample, an antigen can be diluted with 10 mM sodium acetate, pH 4.8, to5 μg/ml (about 0.2 μM) before injection at a flow rate of 5 μl/minute toachieve approximately 10 response units (RU) of coupled protein.Following the injection of antigen, 1 M ethanolamine is injected toblock unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%polysorbate 20 (TWEEN-20®) surfactant (PBST) at at a flow rate ofapproximately 25 μl/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using, for example, a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on) [see, e.g., Chen et al., (1999) J. Mol. Biol.293:865-881]. If the on-rate exceeds, for example, 10⁶M⁻¹ s⁻¹ by thesurface plasmon resonance assay above, then the on-rate can bedetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (e.g.,excitation=295 nm; emission=340 nm, 16 nm band-pass) of a 20 nManti-antigen antibody (Fab form) in PBS in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-AMINCO® spectrophotometer (ThermoSpectronic) with a stirred cuvette.

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g., E. coli or phage), asdescribed herein. The nucleic acid and amino acid sequences of humanGDF11, activin A, activin B, activin C, activin E, GDF8, BMP6, ActRIIB,ALK4, GDF3, and BMP9 are well known in the art. In addition, numerousmethods for generating antibodies are well known in the art, some ofwhich are described herein. Therefore antibody antagonists for use inaccordance with this disclosure may be routinely made by the skilledperson in the art based on the knowledge in the art and teachingsprovided herein.

In certain embodiments, an antibody provided herein is a chimericantibody. A chimeric antibody refers to an antibody in which a portionof the heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species. Certain chimeric antibodies aredescribed, for example, in U.S. Pat. No. 4,816,567; and Morrison et al.,(1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855. In some embodiments, achimeric antibody comprises a non-human variable region (e.g., avariable region derived from a mouse, rat, hamster, rabbit, or non-humanprimate, such as a monkey) and a human constant region. In someembodiments, a chimeric antibody is a “class switched” antibody in whichthe class or subclass has been changed from that of the parent antibody.In general, chimeric antibodies include antigen-binding fragmentsthereof.

In certain embodiments, a chimeric antibody provided herein is ahumanized antibody. A humanized antibody refers to a chimeric antibodycomprising amino acid residues from non-human hypervariable regions(HVRs) and amino acid residues from human framework regions (FRs). Incertain embodiments, a humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the HVRs (e.g., CDRs) correspond to those of anon-human antibody, and all or substantially all of the FRs correspondto those of a human antibody. A humanized antibody optionally maycomprise at least a portion of an antibody constant region derived froma human antibody. A “humanized form” of an antibody, e.g., a non-humanantibody, refers to an antibody that has undergone humanization.Humanized antibodies and methods of making them are reviewed, forexample, in Almagro and Fransson (2008) Front. Biosci. 13:1619-1633 andare further described, for example, in Riechmann et al., (1988) Nature332:323-329; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA86:10029-10033; U.S. Pat. Nos. 5,821,337; 7,527,791; 6,982,321; and U.S.Pat. No. 7,087,409; Kashmiri et al., (2005) Methods 36:25-34 [describingSDR (a-CDR) grafting]; Padlan, Mol. Immunol. (1991) 28:489-498(describing “resurfacing”); Dall'Acqua et al. (2005) Methods 36:43-60(describing “FR shuffling”); Osbourn et al. (2005) Methods 36:61-68; andKlimka et al. Br. J. Cancer (2000) 83:252-260 (describing the “guidedselection” approach to FR shuffling). Human framework regions that maybe used for humanization include but are not limited to: frameworkregions selected using the “best-fit” method [see, e.g., Sims et al.(1993) J. Immunol. 151:2296]; framework regions derived from theconsensus sequence of human antibodies of a particular subgroup of lightor heavy chain variable regions [see, e.g., Carter et al. (1992) Proc.Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol.,151:2623]; human mature (somatically mutated) framework regions or humangermline framework regions [see, e.g., Almagro and Fransson (2008)Front. Biosci. 13:1619-1633]; and framework regions derived fromscreening FR libraries [see, e.g., Baca et al., (1997) J. Biol. Chem.272:10678-10684; and Rosok et al., (1996) J. Biol. Chem.271:22611-22618].

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel (2008) Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr.Opin. Immunol. 20:450-459. For example, human antibodies may be preparedby administering an immunogen (e.g., a GDF11 polypeptide, an activin Bpolypeptide, an ActRIIA polypeptide, or an ActRIIB polypeptide) to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicanimals, the endogenous immunoglobulin loci have generally beeninactivated. For a review of methods for obtaining human antibodies fromtransgenic animals see, for example, Lonberg (2005) Nat. Biotech.23:1117-1125; U.S. Pat. Nos. 6,075,181 and 6,150,584 (describingXENOMOUSE™ technology); U.S. Pat. No. 5,770,429 (describing HuMab®technology); U.S. Pat. No. 7,041,870 (describing K-M MOUSE® technology);and U.S. Patent Application Publication No. 2007/0061900 (describingVelociMouse® technology). Human variable regions from intact antibodiesgenerated by such animals may be further modified, for example, bycombining with a different human constant region.

Human antibodies provided herein can also be made by hybridoma-basedmethods. Human myeloma and mouse-human heteromyeloma cell lines for theproduction of human monoclonal antibodies have been described [see,e.g., Kozbor J. Immunol., (1984) 133: 3001; Brodeur et al. (1987)Monoclonal Antibody Production Techniques and Applications, pp. 51-63,Marcel Dekker, Inc., New York; and Boerner et al. (1991) J. Immunol.,147: 86]. Human antibodies generated via human B-cell hybridomatechnology are also described in Li et al., (2006) Proc. Natl. Acad.Sci. USA, 103:3557-3562. Additional methods include those described, forexample, in U.S. Pat. No. 7,189,826 (describing production of monoclonalhuman IgM antibodies from hybridoma cell lines) and Ni, XiandaiMianyixue (2006) 26(4):265-268 (2006) (describing human-humanhybridomas). Human hybridoma technology (Trioma technology) is alsodescribed in Vollmers and Brandlein (2005) Histol. Histopathol.,20(3):927-937 (2005) and Vollmers and Brandlein (2005) Methods Find Exp.Clin. Pharmacol., 27(3):185-91. Human antibodies provided herein mayalso be generated by isolating Fv clone variable-domain sequencesselected from human-derived phage display libraries. Suchvariable-domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are known in the art and described herein.

For example, antibodies of the present disclosure may be isolated byscreening combinatorial libraries for antibodies with the desiredactivity or activities. A variety of methods are known in the art forgenerating phage display libraries and screening such libraries forantibodies possessing the desired binding characteristics. Such methodsare reviewed, for example, in Hoogenboom et al. (2001) in Methods inMolecular Biology 178:1-37, O'Brien et al., ed., Human Press, Totowa,N.J. and further described, for example, in the McCafferty et al. (1991)Nature 348:552-554; Clackson et al., (1991) Nature 352: 624-628; Markset al. (1992) J. Mol. Biol. 222:581-597; Marks and Bradbury (2003) inMethods in Molecular Biology 248:161-175, Lo, ed., Human Press, Totowa,N.J.; Sidhu et al. (2004) J. Mol. Biol. 338(2):299-310; Lee et al.(2004) J. Mol. Biol. 340(5):1073-1093; Fellouse (2004) Proc. Natl. Acad.Sci. USA 101(34):12467-12472; and Lee et al. (2004) J. Immunol. Methods284(1-2): 119-132.

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al. (1994) Ann. Rev.Immunol., 12: 433-455. Phage typically display antibody fragments,either as single-chain Fv (scFv) fragments or as Fab fragments.Libraries from immunized sources provide high-affinity antibodies to theimmunogen (e.g., GDF11, activin B, ALK4, or ActRIIB) without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned (e.g., from human) to provide a single sourceof antibodies to a wide range of non-self and also self-antigens withoutany immunization as described by Griffiths et al. (1993) EMBO J, 12:725-734. Finally, naive libraries can also be made synthetically bycloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter (1992) J. Mol. Biol., 227: 381-388. Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and U.S. Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

In certain embodiments, an antibody provided herein is a multispecificantibody, for example, a bispecific antibody. Multispecific antibodies(typically monoclonal antibodies) that have binding specificities for atleast two different epitopes (e.g., two, three, four, five, or six ormore) on one or more (e.g., two, three, four, five, six or more)antigens.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulinheavy-chain/light-chain pairs having different specificities [see, e.g.,Milstein and Cuello (1983) Nature 305: 537; International patentpublication no. WO 93/08829; and Traunecker et al. (1991) EMBO J. 10:3655, and U.S. Pat. No. 5,731,168 (“knob-in-hole” engineering)].Multispecific antibodies may also be made by engineering electrostaticsteering effects for making antibody Fc-heterodimeric molecules (see,e.g., WO 2009/089004A1); cross-linking two or more antibodies orfragments [see, e.g., U.S. Pat. No. 4,676,980; and Brennan et al. (1985)Science, 229: 81]; using leucine zippers to produce bispecificantibodies [see, e.g., Kostelny et al. (1992) J. Immunol.,148(5):1547-1553]; using “diabody” technology for making bispecificantibody fragments [see, e.g., Hollinger et al. (1993) Proc. Natl. Acad.Sci. USA, 90:6444-6448]; using single-chain Fv (sFv) dimers [see, e.g.,Gruber et al. (1994) J. Immunol., 152:5368]; and preparing trispecificantibodies (see, e.g., Tutt et al. (1991) J. Immunol. 147: 60.Multispecific antibodies can be prepared as full-length antibodies orantibody fragments. Engineered antibodies with three or more functionalantigen-binding sites, including “Octopus antibodies,” are also includedherein [see, e.g., US 2006/0025576A1].

In certain embodiments, an antibody disclosed herein is a monoclonalantibody. Monoclonal antibody refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical and/or bind the sameepitope, except for possible variant antibodies, e.g., containingnaturally occurring mutations or arising during production of amonoclonal antibody preparation, such variants generally being presentin minor amounts. In contrast to polyclonal antibody preparations, whichtypically include different antibodies directed against differentepitopes, each monoclonal antibody of a monoclonal antibody preparationis directed against a single epitope on an antigen. Thus, the modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present methods may be made by a variety of techniques,including but not limited to the hybridoma method, recombinant DNAmethods, phage-display methods, and methods utilizing transgenic animalscontaining all or part of the human immunoglobulin loci, such methodsand other exemplary methods for making monoclonal antibodies beingdescribed herein.

For example, by using immunogens derived from GDF11,anti-protein/anti-peptide antisera or monoclonal antibodies can be madeby standard protocols [see, e.g., Antibodies: A Laboratory Manual ed. byHarlow and Lane (1988) Cold Spring Harbor Press: 1988]. A mammal, suchas a mouse, hamster, or rabbit, can be immunized with an immunogenicform of the GDF11 polypeptide, an antigenic fragment which is capable ofeliciting an antibody response, or a fusion protein. Techniques forconferring immunogenicity on a protein or peptide include conjugation tocarriers or other techniques well known in the art. An immunogenicportion of a GDF11 polypeptide can be administered in the presence ofadjuvant. The progress of immunization can be monitored by detection ofantibody titers in plasma or serum. Standard ELISA or other immunoassayscan be used with the immunogen as antigen to assess the levels ofantibody production and/or level of binding affinity.

Following immunization of an animal with an antigenic preparation ofGDF11, antisera can be obtained and, if desired, polyclonal antibodiescan be isolated from the serum. To produce monoclonal antibodies,antibody-producing cells (lymphocytes) can be harvested from animmunized animal and fused by standard somatic cell fusion procedureswith immortalizing cells such as myeloma cells to yield hybridoma cells.Such techniques are well known in the art, and include, for example, thehybridoma technique [see, e.g., Kohler and Milstein (1975) Nature, 256:495-497], the human B cell hybridoma technique [see, e.g., Kozbar et al.(1983) Immunology Today, 4:72], and the EBV-hybridoma technique toproduce human monoclonal antibodies [Cole et al. (1985) MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96]. Hybridomacells can be screened immunochemically for production of antibodiesspecifically reactive with a GDF11 polypeptide, and monoclonalantibodies isolated from a culture comprising such hybridoma cells.

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g., a substitution,deletion, and/or addition) at one or more amino acid positions.

For example, the present disclosure contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theantibody in vivo is important yet certain effector functions [e.g.,complement-dependent cytotoxicity (CDC) and antibody-dependent cellularcytotoxicity (ADCC)] are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII FcR expression on hematopoietic cells is summarized in, forexample, Ravetch and Kinet (1991) Annu. Rev. Immunol. 9:457-492.Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest are described in U.S. Pat. No. 5,500,362;Hellstrom, I. et al. (1986) Proc. Natl. Acad. Sci. USA 83:7059-7063];Hellstrom, I et al. (1985) Proc. Natl. Acad. Sci. USA 82:1499-1502; U.S.Pat. No. 5,821,337; Bruggemann, M. et al. (1987) J. Exp. Med.166:1351-1361. Alternatively, non-radioactive assays methods may beemployed (e.g., ACTI™, non-radioactive cytotoxicity assay for flowcytometry; CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay, Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and natural killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, for example, in an animal model such as that disclosed inClynes et al. (1998) Proc. Natl. Acad. Sci. USA 95:652-656. C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity [see, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402]. To assess complementactivation, a CDC assay may be performed [see, e.g, Gazzano-Santoro etal. (1996) J. Immunol. Methods 202:163; Cragg, M. S. et al. (2003) Blood101:1045-1052; and Cragg, M. S, and M. J. Glennie (2004) Blood103:2738-2743]. FcRn binding and in vivo clearance/half-lifedeterminations can also be performed using methods known in the art[see, e.g., Petkova, S. B. et al. (2006) Intl. Immunol.18(12):1759-1769]. Antibodies of the present disclosure with reducedeffector function include those with substitution of one or more of Fcregion residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No.6,737,056). Such Fc mutants include Fc mutants with substitutions at twoor more of amino acid positions 265, 269, 270, 297 and 327, includingthe so-called “DANA” Fc mutant with substitution of residues 265 and 297to alanine (U.S. Pat. No. 7,332,581).

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, for example, inU.S. Pat. No. 7,521,541.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, if an antibody is to be used for bindingan antigen in solution, it may be desirable to test solution binding. Avariety of different techniques are available for testing interactionsbetween antibodies and antigens to identify particularly desirableantibodies. Such techniques include ELISAs, surface plasmon resonancebinding assays (e.g., the Biacore binding assay, Biacore AB, Uppsala,Sweden), sandwich assays (e.g., the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Md.), western blots,immunoprecipitation assays, and immunohistochemistry.

In certain embodiments, amino acid sequence variants of the antibodiesand/or the binding polypeptides provided herein are contemplated. Forexample, it may be desirable to improve the binding affinity and/orother biological properties of the antibody and/or binding polypeptide.Amino acid sequence variants of an antibody and/or binding polypeptidesmay be prepared by introducing appropriate modifications into thenucleotide sequence encoding the antibody and/or binding polypeptide, orby peptide synthesis. Such modifications include, for example, deletionsfrom, and/or insertions into and/or substitutions of residues within theamino acid sequences of the antibody and/or binding polypeptide. Anycombination of deletion, insertion, and substitution can be made toarrive at the final construct, provided that the final constructpossesses the desired characteristics, e.g., target-binding (GDF11and/or activin B binding).

Alterations (e.g., substitutions) may be made in HVRs, for example, toimprove antibody affinity. Such alterations may be made in HVR“hotspots,” i.e., residues encoded by codons that undergo mutation athigh frequency during the somatic maturation process [see, e.g.,Chowdhury (2008) Methods Mol. Biol. 207:179-196 (2008)], and/or SDRs(a-CDRs), with the resulting variant VH or VL being tested for bindingaffinity. Affinity maturation by constructing and reselecting fromsecondary libraries has been described in the art [see, e.g., Hoogenboomet al., in Methods in Molecular Biology 178:1-37, O'Brien et al., ed.,Human Press, Totowa, N.J., (2001). In some embodiments of affinitymaturation, diversity is introduced into the variable genes chosen formaturation by any of a variety of methods (e.g., error-prone PCR, chainshuffling, or oligonucleotide-directed mutagenesis). A secondary libraryis then created. The library is then screened to identify any antibodyvariants with the desired affinity. Another method to introducediversity involves HVR-directed approaches, in which several HVRresidues (e.g., 4-6 residues at a time) are randomized. HVR residuesinvolved in antigen binding may be specifically identified, e.g., usingalanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 inparticular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind to the antigen.For example, conservative alterations (e.g., conservative substitutionsas provided herein) that do not substantially reduce binding affinitymay be made in HVRs. Such alterations may be outside of HVR “hotspots”or SDRs. In certain embodiments of the variant VH and VL sequencesprovided above, each HVR either is unaltered, or contains no more thanone, two or three amino acid substitutions.

A useful method for identification of residues or regions of theantibody and/or the binding polypeptide that may be targeted formutagenesis is called “alanine scanning mutagenesis” as described byCunningham and Wells (1989) Science, 244:1081-1085. In this method, aresidue or group of target residues (e.g., charged residues such as Asp,Arg, His, Lys, and Glu) are identified and replaced by a neutral ornegatively charged amino acid (e.g., alanine or polyalanine) todetermine whether the interaction of the antibody-antigen is affected.Further substitutions may be introduced at the amino acid locationsdemonstrating functional sensitivity to the initial substitutions.Alternatively, or additionally, a crystal structure of anantigen-antibody complex is determined to identify contact pointsbetween the antibody and antigen. Such contact residues and neighboringresidues may be targeted or eliminated as candidates for substitution.Variants may be screened to determine whether they contain the desiredproperties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion of the N- orC-terminus of the antibody to an enzyme (e.g., for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

In certain embodiments, an antibody and/or binding polypeptide providedherein may be further modified to contain additional nonproteinaceousmoieties that are known in the art and readily available. The moietiessuitable for derivatization of the antibody and/or binding polypeptideinclude but are not limited to water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody and/orbinding polypeptide may vary, and if more than one polymer are attached,they can be the same or different molecules. In general, the numberand/or type of polymers used for derivatization can be determined basedon considerations including, but not limited to, the particularproperties or functions of the antibody and/or binding polypeptide to beimproved, whether the antibody derivative and/or binding polypeptidederivative will be used in a therapy under defined conditions.

D. Small Molecule Antagonists

In other aspects, an ALK4:ActRIIB antagonist is a small molecule(ALK4:ActRIIB small molecule antagonist), or combination of smallmolecule antagonists. An ALK4:ActRIIB small molecule antagonist, orcombination of small molecule antagonists, may inhibit, for example, oneor more ALK4:ActRIIB-binding ligands, a type I receptor (e.g., ALK4), atype II receptor (e.g., ActRIIB), and/or co-receptor. In someembodiments, ALK4:ActRIIB small molecule antagonist, or combination ofsmall molecule antagonists, inhibits signaling mediated by one or moreALK4:ActRIIB-binding ligands, for example, as determined in a cell-basedassay such as those described herein. As described herein, ALK4:ActRIIBsmall molecule antagonists may be used, alone or in combination with oneor more supportive therapies or active agents, to treat a patient inneed thereof (e.g., a subject with a bone-related disease or condition,a muscle related disease or condition, or a disease or conditionassociated with excess or unwanted fat).

In some embodiments, an ALK4:ActRIIB small molecule antagonist, orcombination of small molecule antagonists, inhibits at least GDF11. Insome embodiments, an ALK4:ActRIIB small molecule antagonist, orcombination of small molecule antagonists, inhibits at least GDF8. Insome embodiments, an ALK4:ActRIIB small molecule antagonist, orcombination of small molecule antagonists, inhibits at least activin(activin A, activin B, activin C, activin E, activin AB, activin AC,activin BC, activin AE and/or activin BE). In some embodiments, anALK4:ActRIIB small molecule antagonist, or combination of small moleculeantagonists, inhibits at least GDF11, GDF8, and activin. In someembodiments, an ALK4:ActRIIB small molecule antagonist, or combinationof small molecule antagonists, inhibits at least ALK4. In someembodiments, an ALK4:ActRIIB small molecule antagonist, or combinationof small molecule antagonists, inhibits at least ActRIIB In someembodiments, an ALK4:ActRIIB small molecule antagonist, or combinationof small molecule antagonists, inhibits at least BMP6. In someembodiments, an ALK4:ActRIIB small molecule antagonist, or combinationof small molecule antagonists, inhibits at least GDF3. In someembodiments, an ALK4:ActRIIB small molecule antagonist, or combinationof small molecule antagonists, inhibits at least BMP10. In someembodiments, an ALK4:ActRIIB small molecule antagonist, or combinationof small molecule antagonists, as disclosed herein does not inhibit ordoes not substantially inhibit BMP9.

ALK4:ActRIIB small molecule antagonists can be direct or indirectinhibitors. For example, an indirect small molecule antagonist, orcombination of small molecule antagonists, may inhibit the expression(e.g., transcription, translation, cellular secretion, or combinationsthereof) of at least one or more TGF-β superfamily ligands that bind toan ALK4:ActRIIB heteromultimer [e.g., activin (e.g., activin A, activinB, and activin AB), GDF8, GDF11, BMP10, BMP6, and GDF3], type I receptor(e.g., ALK4), type II receptors (e.g., ActRIIB), and/or one or moredownstream signaling components (e.g., Smads). Alternatively, a directsmall molecule antagonist, or combination of small molecule antagonists,may directly bind to and inhibit, for example, one or more TGF-βsuperfamily ligands that bind to an ALK4:ActRIIB heteromultimer [e.g.,activin (e.g., activin A, activin B, and activin AB), GDF11, GDF8,BMP10, BMP6, and GDF3], type I receptors (e.g., ALK4), type II receptors(e.g., ActRIIB), co-receptors (e.g., Cripto or Cryptic), and/ordownstream signaling components (e.g., Smads). Combinations of one ormore indirect and one or more direct ALK4:ActRIIB small moleculeantagonists may be used in accordance with the methods disclosed herein.

Binding small-molecule antagonists of the present disclosure may beidentified and chemically synthesized using known methodology (see,e.g., PCT Publication Nos. WO 00/00823 and WO 00/39585). In general,small-molecule antagonists of the disclosure are usually less than about2000 daltons in size, alternatively less than about 1500, 750, 500, 250or 200 daltons in size, wherein such organic small molecules that arecapable of binding, preferably specifically, to a polypeptide asdescribed herein. These small molecule antagonists may be identifiedwithout undue experimentation using well-known techniques. In thisregard, it is noted that techniques for screening organic small-moleculelibraries for molecules that are capable of binding to a polypeptidetarget are well known in the art (see, e.g., international patentpublication Nos. WO00/00823 and WO00/39585).

Binding organic small molecules of the present disclosure may be, forexample, aldehydes, ketones, oximes, hydrazones, semicarbazones,carbazides, primary amines, secondary amines, tertiary amines,N-substituted hydrazines, hydrazides, alcohols, ethers, thiols,thioethers, disulfides, carboxylic acids, esters, amides, ureas,carbamates, carbonates, ketals, thioketals, acetals, thioacetals, arylhalides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromaticcompounds, heterocyclic compounds, anilines, alkenes, alkynes, diols,amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines,enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonylchlorides, diazo compounds, and acid chlorides.

E. Polynucleotide Antagonists

In other aspects, an ALK4:ActRIIB antagonist is a polynucleotide(ALK4:ActRIIB polynucleotide antagonist), or combination ofpolynucleotides. An ALK4:ActRIIB polynucleotide antagonist, orcombination of polynucleotide antagonists, may inhibit, for example, oneor more ALK4:ActRIIB-binding ligands [e.g., activin (e.g., activin A,activin B, and activin AB), GDF8, GDF11, BMP10, BMP6, and GDF3], type Ireceptors (e.g., ALK4), type II receptors (e.g., ActRIIB), co-receptor,and/or downstream signaling component (e.g., Smads). In someembodiments, ALK4:ActRIIB polynucleotide antagonist, or combination ofpolynucleotide antagonists, inhibits signaling mediated by one or moreALK4:ActRIIB-binding ligands, for example, as determined in a cell-basedassay such as those described herein. As described herein, ALK4:ActRIIBpolynucleotide antagonists may be used, alone or in combination with oneor more supportive therapies or active agents, to treat a patient inneed thereof (e.g., a subject with a bone-related disease or condition,a muscle related disease or condition, or a disease or conditionassociated with excess or unwanted fat).

In some embodiments, an ALK4:ActRIIB polynucleotide antagonists, orcombination of polynucleotide antagonists, inhibits at least GDF11. Insome embodiments, an ALK4:ActRIIB polynucleotide antagonist, orcombination of polynucleotide antagonists, inhibits at least GDF8. Insome embodiments, an ALK4:ActRIIB polynucleotide antagonist, orcombination of polynucleotide antagonists, inhibits at least activin(activin A, activin B, activin C, activin E, activin AB, activin AC,activin AE, activin BC and/or activin BE). In some embodiments, anALK4:ActRIIB polynucleotide antagonist, or combination of polynucleotideantagonists, inhibits at least GDF11, GDF8, and activin. In someembodiments, an ALK4:ActRIIB polynucleotide antagonist, or combinationof polynucleotide antagonists, inhibits at least ALK4. In someembodiments, an ALK4:ActRIIB polynucleotide antagonist, or combinationof polynucleotide antagonists, inhibits at least ActRIIB In someembodiments, an ALK4:ActIIB polynucleotide antagonist, or combination ofpolynucleotide antagonists, inhibits at least BMP6. In some embodiments,an ALK4:ActRIIB polynucleotide antagonist, or combination ofpolynucleotide antagonists, inhibits at least GDF3. In some embodiments,an ALK4:ActRIIB polynucleotide antagonist, or combination ofpolynucleotide antagonists, inhibits at least BMP10. In someembodiments, an ALK4:ActRIIB polynucleotide antagonist, or combinationof polynucleotide antagonists, as disclosed herein does not inhibit ordoes not substantially inhibit BMP9.

In some embodiments, the polynucleotide antagonists of the disclosuremay be an antisense nucleic acid, an RNAi molecule [e.g., smallinterfering RNA (siRNA), small-hairpin RNA (shRNA), microRNA (miRNA)],an aptamer and/or a ribozyme. The nucleic acid and amino acid sequencesof human GDF11, activin B, GDF8, activin A, BMP6, GDF3, ALK4, ActRIIB,and BMP10 are known in the art. In addition, many different methods ofgenerating polynucleotide antagonists are well known in the art.Therefore polynucleotide antagonists for use in accordance with thisdisclosure may be routinely made by the skilled person in the art basedon the knowledge in the art and teachings provided herein.

Antisense technology can be used to control gene expression throughantisense DNA or RNA, or through triple-helix formation. Antisensetechniques are discussed, for example, in Okano (1991) J. Neurochem.56:560; Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple-helix formationis discussed in, for instance, Cooney et al. (1988) Science 241:456; andDervan et al., (1991) Science 251:1300. The methods are based on bindingof a polynucleotide to a complementary DNA or RNA. In some embodiments,the antisense nucleic acids comprise a single-stranded RNA or DNAsequence that is complementary to at least a portion of an RNAtranscript of a gene disclosed herein. However, absolutecomplementarity, although preferred, is not required.

A sequence “complementary to at least a portion of an RNA,” referred toherein, means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex; in the case ofdouble-stranded antisense nucleic acids of a gene disclosed herein, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acid.Generally, the larger the hybridizing nucleic acid, the more basemismatches with an RNA it may contain and still form a stable duplex (ortriplex as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

Polynucleotides that are complementary to the 5′ end of the message, forexample, the 5′-untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′-untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well [see, e.g., Wagner, R., (1994) Nature372:333-335]. Thus, oligonucleotides complementary to either the 5′- or3′-non-translated, non-coding regions of a gene of the disclosure, couldbe used in an antisense approach to inhibit translation of an endogenousmRNA. Polynucleotides complementary to the 5′-untranslated region of themRNA should include the complement of the AUG start codon. Antisensepolynucleotides complementary to mRNA coding regions are less efficientinhibitors of translation but could be used in accordance with themethods of the present disclosure. Whether designed to hybridize to the5′-, 3′- or coding region of an mRNA of the disclosure, antisensenucleic acids should be at least six nucleotides in length, and arepreferably oligonucleotides ranging from 6 to about 50 nucleotides inlength. In specific aspects the oligonucleotide is at least 10nucleotides, at least 17 nucleotides, at least 25 nucleotides or atleast 50 nucleotides.

In one embodiment, the antisense nucleic acid of the present disclosureis produced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof is transcribed, producing anantisense nucleic acid (RNA) of a gene of the disclosure. Such a vectorwould contain a sequence encoding the desired antisense nucleic acid.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding desired genes of the instantdisclosure, or fragments thereof, can be by any promoter known in theart to act in vertebrate, preferably human cells. Such promoters can beinducible or constitutive. Such promoters include, but are not limitedto, the SV40 early promoter region [see, e.g., Benoist and Chambon(1981) Nature 290:304-310], the promoter contained in the 3′long-terminal repeat of Rous sarcoma virus [see, e.g., Yamamoto et al.(1980) Cell 22:787-797], the herpes thymidine promoter [see, e.g.,Wagner et al. (1981) Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445], andthe regulatory sequences of the metallothionein gene [see, e.g.,Brinster, et al. (1982) Nature 296:39-42].

In some embodiments, the polynucleotide antagonists are interfering RNA(RNAi) molecules that target the expression of one or more of: GDF11,activin B, GDF8, activin A, BMP6, GDF3, BMP10, ALK4, and ActRIIB RNAirefers to the expression of an RNA which interferes with the expressionof the targeted mRNA. Specifically, RNAi silences a targeted gene viainteracting with the specific mRNA through a siRNA (small interferingRNA). The ds RNA complex is then targeted for degradation by the cell.An siRNA molecule is a double-stranded RNA duplex of 10 to 50nucleotides in length, which interferes with the expression of a targetgene which is sufficiently complementary (e.g. at least 80% identity tothe gene). In some embodiments, the siRNA molecule comprises anucleotide sequence that is at least 85, 90, 95, 96, 97, 98, 99, or 100%identical to the nucleotide sequence of the target gene.

Additional RNAi molecules include short-hairpin RNA (shRNA); alsoshort-interfering hairpin and microRNA (miRNA). The shRNA moleculecontains sense and antisense sequences from a target gene connected by aloop. The shRNA is transported from the nucleus into the cytoplasm, andit is degraded along with the mRNA. Pol III or U6 promoters can be usedto express RNAs for RNAi. Paddison et al. [Genes & Dev. (2002)16:948-958, 2002] have used small RNA molecules folded into hairpins asa means to affect RNAi. Accordingly, such short-hairpin RNA (shRNA)molecules are also advantageously used in the methods described herein.The length of the stem and loop of functional shRNAs varies; stemlengths can range anywhere from about 25 to about 30 nt, and loop sizecan range between 4 to about 25 nt without affecting silencing activity.While not wishing to be bound by any particular theory, it is believedthat these shRNAs resemble the double-stranded RNA (dsRNA) products ofthe DICER RNase and, in any event, have the same capacity for inhibitingexpression of a specific gene. The shRNA can be expressed from alentiviral vector. An miRNA is a single-stranded RNA of about 10 to 70nucleotides in length that are initially transcribed as pre-miRNAcharacterized by a “stem-loop” structure, which are subsequentlyprocessed into mature miRNA after further processing through the RISC.

Molecules that mediate RNAi, including without limitation siRNA, can beproduced in vitro by chemical synthesis (Hohjoh, FEBS Lett 521:195-199,2002), hydrolysis of dsRNA (Yang et al., Proc Natl Acad Sci USA99:9942-9947, 2002), by in vitro transcription with T7 RNA polymerase(Donzeet et al., Nucleic Acids Res 30:e46, 2002; Yu et al., Proc NatlAcad Sci USA 99:6047-6052, 2002), and by hydrolysis of double-strandedRNA using a nuclease such as E. coli RNase III (Yang et al., Proc NatlAcad Sci USA 99:9942-9947, 2002).

According to another aspect, the disclosure provides polynucleotideantagonists including but not limited to, a decoy DNA, a double-strandedDNA, a single-stranded DNA, a complexed DNA, an encapsulated DNA, aviral DNA, a plasmid DNA, a naked RNA, an encapsulated RNA, a viral RNA,a double-stranded RNA, a molecule capable of generating RNAinterference, or combinations thereof.

In some embodiments, the polynucleotide antagonists of the disclosureare aptamers. Aptamers are nucleic acid molecules, includingdouble-stranded DNA and single-stranded RNA molecules, which bind to andform tertiary structures that specifically bind to a target molecule.The generation and therapeutic use of aptamers are well established inthe art (see, e.g., U.S. Pat. No. 5,475,096). Additional information onaptamers can be found in U.S. Patent Application Publication No.20060148748. Nucleic acid aptamers are selected using methods known inthe art, for example via the Systematic Evolution of Ligands byExponential Enrichment (SELEX) process. SELEX is a method for the invitro evolution of nucleic acid molecules with highly specific bindingto target molecules as described in, e.g., U.S. Pat. Nos. 5,475,096;5,580,737; 5,567,588; 5,707,796; 5,763,177; 6,011,577; and 6,699,843.Another screening method to identify aptamers is described in U.S. Pat.No. 5,270,163. The SELEX process is based on the capacity of nucleicacids for forming a variety of two- and three-dimensional structures, aswell as the chemical versatility available within the nucleotidemonomers to act as ligands (form specific binding pairs) with virtuallyany chemical compound, whether monomeric or polymeric, including othernucleic acid molecules and polypeptides. Molecules of any size orcomposition can serve as targets. The SELEX method involves selectionfrom a mixture of candidate oligonucleotides and step-wise iterations ofbinding, partitioning and amplification, using the same generalselection scheme, to achieve desired binding affinity and selectivity.Starting from a mixture of nucleic acids, which can comprise a segmentof randomized sequence, the SELEX method includes steps of contactingthe mixture with the target under conditions favorable for binding;partitioning unbound nucleic acids from those nucleic acids which havebound specifically to target molecules; dissociating the nucleicacid-target complexes; amplifying the nucleic acids dissociated from thenucleic acid-target complexes to yield a ligand enriched mixture ofnucleic acids. The steps of binding, partitioning, dissociating andamplifying are repeated through as many cycles as desired to yieldnucleic acid ligands which bind with high affinity and specificity tothe target molecule.

Typically, such binding molecules are separately administered to theanimal [see, e.g., O'Connor (1991) J. Neurochem. 56:560], but suchbinding molecules can also be expressed in vivo from polynucleotidestaken up by a host cell and expressed in vivo [see, e.g.,Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)].

F. Follistatin and FLRG Antagonists

It is known that members of the follistatin and FLRG group of proteinsantagonize ligands that signal through the ALK4:ActRIIB pathway.Accordingly, in other aspects, an ALK4:ActRIIB antagonist is afollistatin or FLRG polypeptide, which may be used alone or incombination with one or more additional supportive therapies and/oractive agents as disclosed herein to achieve a desired effect (e.g.,treat patients having kidney disease and/or a metabolic disorder).

The term “follistatin polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of follistatin as well as any variantsthereof (including mutants, fragments, fusions, and peptidomimeticforms) that retain a useful activity, and further includes anyfunctional monomer or multimer of follistatin. In certain preferredembodiments, follistatin polypeptides of the disclosure bind to and/orinhibit activin and/or GDF8 activity. Variants of follistatinpolypeptides that retain activin binding properties can be identifiedbased on previous studies involving follistatin and activininteractions. For example, WO2008/030367 discloses specific follistatindomains (“FSDs”) that are shown to be important for activin binding. Asshown below in SEQ ID NOs: 90-94, the follistatin N-terminal domain(“FSND” SEQ ID NO: 92), FSD2 (SEQ ID NO: 94), and to a lesser extentFSD1 (SEQ ID NO: 93) represent exemplary domains within follistatin thatare important for activin binding. In addition, methods for making andtesting libraries of polypeptides are described above in the context ofActRII polypeptides, and such methods also pertain to making and testingvariants of follistatin. Follistatin polypeptides include polypeptidesderived from the sequence of any known follistatin having a sequence atleast about 80% identical to the sequence of a follistatin polypeptide,and optionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or greateridentity. Examples of follistatin polypeptides include the maturefollistatin polypeptide or shorter isoforms or other variants of thehuman follistatin precursor polypeptide (SEQ ID NO: 90) as described,for example, in WO2005/025601.

The human follistatin precursor polypeptide isoform FST344 is asfollows:

(SEQ ID NO: 90; NCBI Reference No. NP_037541.1)  1 MVRARHQPGG LCLLLLLLCQ FMEDRSAQAG NCWLRQAKNG RCQVLYKTEL 51 SKEECCSTGR LSTSWTEEDV NDNTLFKWMI FNGGAPNCIP CKETCENVDC101 GPGKKCRMNK KNKPRCVCAP DCSNITWKGP VCGLDGKTYR NECALLKARC151 KEQPELEVQY QGRCKKTCRD VFCPGSSTCV VDQTNNAYCV TCNRICPEPA201 SSEQYLCGND GVTYSSACHL RKATCLLGRS IGLAYEGKCI KAKSCEDIQC251 TGGKKCLWDF KVGRGRCSLC DELCPDSKSD EPVCASDNAT YASECAMKEA301 ACSSGVLLEV KHSGSCNSIS EDTEEEEEDE DQDYSFPISS ILEW

The signal peptide is underlined; also underlined above are the last 27residues which represent the C-terminal extension distinguishing thisfollistatin isoform from the shorter follistatin isoform FST317 shownbelow.

The human follistatin precursor polypeptide isoform FST317 is asfollows:

(SEQ ID NO: 91; NCBI Reference No. NP_006341.1)  1 MVRARHQPGG LCLLLLLLCQ FMEDRSAQAG NCWLRQAKNG RCQVLYKTEL 51 SKEECCSTGR LSTSWTEEDV NDNTLFKWMI FNGGAPNCIP CKETCENVDC101 GPGKKCRMNK KNKPRCVCAP DCSNITWKGP VCGLDGKTYR NECALLKARC151 KEQPELEVQY QGRCKKTCRD VFCPGSSTCV VDQTNNAYCV TCNRICPEPA201 SSEQYLCGND GVTYSSACHL RKATCLLGRS IGLAYEGKCI KAKSCEDIQC251 TGGKKCLWDF KVGRGRCSLC DELCPDSKSD EPVCASDNAT YASECAMKEA301 ACSSGVLLEV KHSGSCNThe signal peptide is underlined.

The follistatin N-terminal domain (FSND) sequence is as follows:

(SEQ ID NO: 92; FSND) GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCK

The FSD1 and FSD2 sequences are as follows:

(SEQ ID NO: 93; FSD1) ETCENVDCGPGKKCRMNKKNKPRCV (SEQ ID NO: 94; FSD2)KTCRDVFCPGSSTCVVDQTNNAYCVT

In other aspects, an ALK4:ActRIIB antagonist is a follistatin-likerelated gene (FLRG), also known as follistatin-related protein 3(FSTL3). The term “FLRG polypeptide” includes polypeptides comprisingany naturally occurring polypeptide of FLRG as well as any variantsthereof (including mutants, fragments, fusions, and peptidomimeticforms) that retain a useful activity. In certain embodiments, FLRGpolypeptides of the disclosure bind to and/or inhibit activin activity,particularly activin A. Variants of FLRG polypeptides that retainactivin binding properties can be identified using routine methods toassay FLRG and activin interactions (see, e.g., U.S. Pat. No.6,537,966). In addition, methods for making and testing libraries ofpolypeptides are described above in the context of ActRII and ALK4polypeptides and such methods also pertain to making and testingvariants of FLRG. FLRG polypeptides include polypeptides derived fromthe sequence of any known FLRG having a sequence at least about 80%identical to the sequence of an FLRG polypeptide, and optionally atleast 85%, 90%, 95%, 97%, 99% or greater identity.

The human FLRG precursor (follistatin-related protein 3 precursor)polypeptide is as follows:

(SEQ ID NO: 95; NCBI Reference No. NP_005851.1)  1 MRPGAPGPLW PLPWGALAWA VGFVSSMGSG NPAPGGVCWL QQGQEATCSL 51 VLQTDVTRAE CCASGNIDTA WSNLTHPGNK INLLGFLGLV HCLPCKDSCD101 GVECGPGKAC RMLGGRPRCE CAPDCSGLPA RLQVCGSDGA TYRDECELRA151 ARCRGHPDLS VMYRGRCRKS CEHVVCPRPQ SCVVDQTGSA HCVVCRAAPC201 PVPSSPGQEL CGNNNVTYIS SCHMRQATCF LGRSIGVRHA GSCAGTPEEP251 PGGESAEEEE NFVThe signal peptide is underlined.

In certain embodiments, functional variants or modified forms of thefollistatin polypeptides and FLRG polypeptides include fusion proteinshaving at least a portion of the follistatin polypeptide or FLRGpolypeptide and one or more fusion domains, such as, for example,domains that facilitate isolation, detection, stabilization ormultimerization of the polypeptide. Suitable fusion domains arediscussed in detail above with reference to the ActRII polypeptides. Insome embodiment, an antagonist agent of the disclosure is a fusionprotein comprising an activin-binding portion of a follistatinpolypeptide fused to an Fc domain. In another embodiment, an antagonistagent of the disclosure is a fusion protein comprising an activinbinding portion of an FLRG polypeptide fused to an Fc domain.

5. Screening Assays

In certain aspects, the present disclosure relates to the use ofALK4:ActRIIB heteromultimers to identify compounds (agents) which areagonists or antagonists of TGFβ superfamily receptors. Compoundsidentified through this screening can be tested to assess their abilityto modulate tissues such as bone, cartilage, muscle, fat, and/orneurons, to assess their ability to modulate tissue growth in vivo or invitro. These compounds can be tested, for example, in animal models.

There are numerous approaches to screening for therapeutic agents formodulating tissue growth by targeting TGFβ superfamily ligand signaling(e.g., SMAD 2/3 and/or SMAD 1/5/8 signaling). In certain embodiments,high-throughput screening of compounds can be carried out to identifyagents that perturb TGFβ superfamily receptor-mediated effects on aselected cell line. In certain embodiments, the assay is carried out toscreen and identify compounds that specifically inhibit or reducebinding of an ALK4:ActRIIB heteromultimer to its binding partner, suchas a TGFβ superfamily ligand (e.g., BMP2, BMP2/7, BMP3, BMP4, BMP4/7,BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5, GDF6/BMP13,GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1, TGF-β1, TGF-β2,TGF-β3, activin A, activin B, activin AB, activin AC, nodal, glialcell-derived neurotrophic factor (GDNF), neurturin, artemin, persephin,MIS, and Lefty). Alternatively, the assay can be used to identifycompounds that enhance binding of an ALK4:ActRIIB heteromultimer to itsbinding partner such as an TGFβ superfamily ligand. In a furtherembodiment, the compounds can be identified by their ability to interactwith an ALK4:ActRIIB heteromultimers.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof tissue growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. Incertain embodiments, the test agent is a small organic molecule having amolecular weight of less than about 2,000 Daltons.

The test compounds of the disclosure can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S-transferase (GST),photoactivatible crosslinkers or any combinations thereof.

In many drug-screening programs which test libraries of compounds andnatural extracts, high-throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between anALK4:ActRIIB heteromultimers and its binding partner (e.g., BMP2,BMP2/7, BMP3, BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10,GDF3, GDF5, GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11,GDF15/MIC1, TGF-β1, TGF-β2, TGF-β3, activin A, activin B, activin C,activin E, activin AB, activin AC, nodal, glial cell-derivedneurotrophic factor (GDNF), neurturin, artemin, persephin, MIS, andLefty).

Merely to illustrate, in an exemplary screening assay of the presentdisclosure, the compound of interest is contacted with an isolated andpurified ALK4:ActRIIB heteromultimers which is ordinarily capable ofbinding to a TGF-beta superfamily ligand, as appropriate for theintention of the assay. To the mixture of the compound and ALK4:ActRIIBheteromultimer is then added to a composition containing the appropriateTGF-beta superfamily ligand (e.g., BMP2, BMP2/7, BMP3, BMP4, BMP4/7,BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5, GDF6/BMP13,GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1, TGF-β1, TGF-β2,TGF-β3, activin A, activin B, activin C, activin E, activin AB, activinAC, nodal, glial cell-derived neurotrophic factor (GDNF), neurturin,artemin, persephin, MIS, and Lefty). Detection and quantification ofheteromultimer-superfamily ligand complexes provides a means fordetermining the compound's efficacy at inhibiting (or potentiating)complex formation between the ALK4:ActRIIB heteromultimer and itsbinding protein. The efficacy of the compound can be assessed bygenerating dose-response curves from data obtained using variousconcentrations of the test compound. Moreover, a control assay can alsobe performed to provide a baseline for comparison. For example, in acontrol assay, isolated and purified TGF-beta superfamily ligand isadded to a composition containing the ALK4:ActRIIB heteromultimer, andthe formation of heteromultimer-ligand complex is quantitated in theabsence of the test compound. It will be understood that, in general,the order in which the reactants may be admixed can be varied, and canbe admixed simultaneously. Moreover, in place of purified proteins,cellular extracts and lysates may be used to render a suitable cell-freeassay system.

Binding of a ALK4:ActRIIB heteromultimer to another protein may bedetected by a variety of techniques. For instance, modulation of theformation of complexes can be quantitated using, for example, detectablylabeled proteins such as radiolabeled (e.g., ³²P, ³⁵S, ¹⁴C or ³H),fluorescently labeled (e.g., FITC), or enzymatically labeledALK4:ActRIIB heteromeric and/or its binding protein, by immunoassay, orby chromatographic detection.

In certain embodiments, the present disclosure contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between an ALK4:ActRIIB heteromultimer and itsbinding protein. Further, other modes of detection, such as those basedon optical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No.5,677,196), surface plasmon resonance (SPR), surface charge sensors, andsurface force sensors, are compatible with many embodiments of thedisclosure.

Moreover, the present disclosure contemplates the use of an interactiontrap assay, also known as the “two-hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between an ALK4:ActRIIBheteromultimer and its binding partner. See, e.g., U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) JBiol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696). In aspecific embodiment, the present disclosure contemplates the use ofreverse two-hybrid systems to identify compounds (e.g., small moleculesor peptides) that dissociate interactions between an ALK4:ActRIIBheteromultimer and its binding protein [Vidal and Legrain, (1999)Nucleic Acids Res 27:919-29; Vidal and Legrain, (1999) Trends Biotechnol17:374-81; and U.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368].

In certain embodiments, the subject compounds are identified by theirability to interact with an ALK4:ActRIIB heteromultimer of thedisclosure. The interaction between the compound and the ALK4:ActRIIBheteromultimer may be covalent or non-covalent. For example, suchinteraction can be identified at the protein level using in vitrobiochemical methods, including photo-crosslinking, radiolabeled ligandbinding, and affinity chromatography [Jakoby W B et al. (1974) Methodsin Enzymology 46:1]. In certain cases, the compounds may be screened ina mechanism-based assay, such as an assay to detect compounds which bindto an ALK4:ActRIIB heteromultime. This may include a solid-phase orfluid-phase binding event. Alternatively, the gene encoding anALK4:ActRIIB heteromultimer can be transfected with a reporter system(e.g., β-galactosidase, luciferase, or green fluorescent protein) into acell and screened against the library preferably by high-throughputscreening or with individual members of the library. Othermechanism-based binding assays may be used; for example, binding assayswhich detect changes in free energy. Binding assays can be performedwith the target fixed to a well, bead or chip or captured by animmobilized antibody or resolved by capillary electrophoresis. The boundcompounds may be detected usually using colorimetric endpoints orfluorescence or surface plasmon resonance.

6. Exemplary Therapeutic Uses

In certain embodiments, an ALK4:ActRIIB antagonist, or combinations ofALK4:ActRIIB antagonists, of the present disclosure can be used to treator prevent a disease or condition that is associated with abnormalactivity of an ALK4:ActRIIB-binding ligand. These diseases, disorders,or conditions are generally referred to herein as“ALK4:ActRIIB-associated conditions” or “ALK4:ActRIIB-associateddisorders.” In certain embodiments, the present disclosure providesmethods of treating or preventing an ALK4:ActRIIB-associated conditionin an individual by administering to an individual in need thereof atherapeutically effective amount of an ALK4:ActRIIB antagonist (e.g., anALK4:ActRIIB heteromultimer such as an ALK4:ActRIIB heterodimer), orcombinations of such antagonists, as described herein. The terms“subject,” an “individual,” or a “patient” are interchangeablethroughout the specification. Any of the ALK4:ActRIIB antagonists of thedisclosure can potentially be employed individually or in combinationfor therapeutic uses disclosed herein. These methods are particularlyaimed at therapeutic and prophylactic treatments of mammals including,for example, rodents, primates, and humans.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes amelioration or elimination of the condition once it has beenestablished. In either case, prevention or treatment may be discerned inthe diagnosis provided by a physician or other health care provider andthe intended result of administration of the therapeutic agent.

In general, treatment or prevention of a disease or condition asdescribed in the present disclosure is achieved by administering anALK4:ActRIIB antagonist, or combinations of such antagonists, of thepresent disclosure in an “effective amount”. An effective amount of anagent refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result. A“therapeutically effective amount” of an agent of the present disclosuremay vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the agent to elicit adesired response in the individual. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result.

Naturally occurring ALK4 and ActRIIB receptor-ligand complexes playessential roles in tissue growth as well as early developmentalprocesses such as the correct formation of various structures or in oneor more post-developmental capacities including sexual development,pituitary hormone production, and creation of bone and cartilage. Thus,ALK4:ActRIIB-associated conditions include, but are not limited to,abnormal tissue growth and developmental defects. In addition,ALK4:ActRIIB-associated conditions include, but are not limited to,disorders of cell growth and differentiation such as inflammation,allergy, autoimmune diseases, and tumors.

For example, ALK4:ActRIIB-associated conditions include neuromusculardisorders (e.g., muscular dystrophy and muscle atrophy), congestiveobstructive pulmonary disease (and muscle wasting associated with COPD),muscle wasting syndrome, sarcopenia, cachexia, adipose tissue disorders(e.g., obesity), type 2 diabetes (NIDDM, adult-onset diabetes), and bonedegenerative disease (e.g., osteoporosis). Other exemplaryALK4:ActRIIB-associated conditions include musculodegenerative andneuromuscular disorders, tissue repair (e.g., wound healing),neurodegenerative diseases (e.g., amyotrophic lateral sclerosis), andimmunologic disorders (e.g., disorders related to abnormal proliferationor function of lymphocytes).

In certain embodiments, an ALK4:ActRIIB antagonist (e.g., anALK4:ActRIIB heterodimer), or combinations of such antagonists, of thedisclosure may be used as part of a treatment for a muscular dystrophy.The term “muscular dystrophy” refers to a group of degenerative musclediseases characterized by gradual weakening and deterioration ofskeletal muscles and sometimes the heart and respiratory muscles.Muscular dystrophies are genetic disorders characterized by progressivemuscle wasting and weakness that begin with microscopic changes in themuscle. As muscles degenerate over time, the person's muscle strengthdeclines. Exemplary muscular dystrophies that can be treated with aregimen including the subject TGF-beta superfamily heteromultimercomplexes include: Duchenne muscular dystrophy (DMD), Becker musculardystrophy (BMD), Emery-Dreifuss muscular dystrophy (EDMD), limb-girdlemuscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSHor FSHD) (also known as Landouzy-Dejerine), myotonic dystrophy (MMD;also known as Steinert's Disease), oculopharyngeal muscular dystrophy(OPMD), distal muscular dystrophy (DD), congenital muscular dystrophy(CMD).

Duchenne muscular dystrophy (DMD) was first described by the Frenchneurologist Guillaume Benjamin Amand Duchenne in the 1860s. Beckermuscular dystrophy (BMD) is named after the German doctor Peter EmilBecker, who first described this variant of DMD in the 1950s. DMD is oneof the most frequent inherited diseases in males, affecting one in 3,500boys. DMD occurs when the dystrophin gene, located on the short arm ofthe X chromosome, is defective. Since males only carry one copy of the Xchromosome, they only have one copy of the dystrophin gene. Without thedystrophin protein, muscle is easily damaged during cycles ofcontraction and relaxation. While early in the disease musclecompensates by regeneration, later on muscle progenitor cells cannotkeep up with the ongoing damage and healthy muscle is replaced bynon-functional fibro-fatty tissue.

BMD results from different mutations in the dystrophin gene. BMDpatients have some dystrophin, but it is either of insufficient quantityor poor quality. The presence of some dystrophin protects the muscles ofpatients with BMD from degenerating as severely or as quickly as thoseof patients with DMD.

Studies in animals indicate that inhibition of the GDF8 signalingpathway may effectively treat various aspects of disease in DMD and BMDpatients (Bogdanovich et al., 2002, Nature 420:418-421; Pistilli et al.,2011, Am J Pathol 178:1287-1297). Thus, ALK4:ActRIIB antagonists of thedisclosure may act as GDF8 inhibitors (antagonists), and constitute analternative means of blocking signaling by GDF8 and/or related TGFβsuperfamily ligands in vivo in DMD and BMD patients.

Similarly, ALK4:ActRIIB antagonists of the disclosure may provide aneffective means to increase muscle mass in other disease conditions thatare in need of muscle growth. For example, amyotrophic lateral sclerosis(ALS), also called Lou Gehrig's disease or motor neuron disease, is achronic, progressive, and incurable CNS disorder that attacks motorneurons, which are components of the central nervous system required forinitiation of skeletal muscle contraction. In ALS, motor neuronsdeteriorate and eventually die, and though a person's brain normallyremains fully functioning and alert, initiation of muscle contraction isblocked at the spinal level. Individuals who develop ALS are typicallybetween 40 and 70 years old, and the first motor neurons to degenerateare those innervating the arms or legs. Patients with ALS may havetrouble walking, may drop things, fall, slur their speech, and laugh orcry uncontrollably. As the disease progresses, muscles in the limbsbegin to atrophy from disuse. Muscle weakness becomes debilitating, andpatients eventually require a wheel chair or become confined to bed.Most ALS patients die from respiratory failure or from complications ofventilator assistance like pneumonia 3-5 years from disease onset.

Promotion of increased muscle mass by ALK4:ActRIIB antagonists mightalso benefit those suffering from muscle wasting diseases.Gonzalez-Cadavid et al. (supra) reported that GDF8 expression correlatesinversely with fat-free mass in humans and that increased expression ofthe GDF8 gene is associated with weight loss in men with AIDS wastingsyndrome. By inhibiting the function of GDF8 in AIDS patients, at leastcertain symptoms of AIDS may be alleviated, if not completelyeliminated, thus significantly improving quality of life in AIDSpatients.

Since loss of GDF8 function is also associated with fat loss withoutdiminution of nutrient intake (Zimmers et al., supra; McPherron and Lee,supra), the subject ALK4:ActRIIB antagonists may further be used as atherapeutic agent for slowing or preventing the development of obesityand type 2 diabetes.

Cancer anorexia-cachexia syndrome is among the most debilitating andlife-threatening aspects of cancer. This syndrome is a common feature ofmany types of cancer—present in approximately 80% of cancer patients atdeath—and is responsible not only for a poor quality of life and poorresponse to chemotherapy but also a shorter survival time than is foundin patients with comparable tumors but without weight loss. Cachexia istypically suspected in patients with cancer if an involuntary weightloss of greater than five percent of premorbid weight occurs within asix-month period. Associated with anorexia, wasting of fat and muscletissue, and psychological distress, cachexia arises from a complexinteraction between the cancer and the host. Cancer cachexia affectscytokine production, release of lipid-mobilizing andproteolysis-inducing factors, and alterations in intermediarymetabolism. Although anorexia is common, a decreased food intake aloneis unable to account for the changes in body composition seen in cancerpatients, and increasing nutrient intake is unable to reverse thewasting syndrome. Currently, there is no treatment to control or reversethe cachexic process. Since systemic overexpression of GDF8 in adultmice was found to induce profound muscle and fat loss analogous to thatseen in human cachexia syndromes (Zimmers et al., supra), the subjectALK4:ActRIIB antagonists may be beneficially used to prevent, treat, oralleviate the symptoms of the cachexia syndrome, where muscle growth isdesired. An example of a heteromeric complex useful for preventing,treating, or alleviating muscle loss as described above is anALK4:ActRIIB heterodimer.

In certain embodiments, an ALK4:ActRIIB antagonist (e.g., anALK4:ActrIIB heterodimer), or combinations of such antagonists, of thepresent disclosure may be used in methods of inducing bone and/orcartilage formation, preventing bone loss, increasing bonemineralization, preventing the demineralization of bone, and/orincreasing bone density. ALK4:ActRIIB antagonists may be useful inpatients who are diagnosed with subclinical low bone density, as aprotective measure against the development of osteoporosis.

In some embodiments, an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIBheterodimer), or combinations of such antagonists, of the presentdisclosure may find medical utility in the healing of bone fractures andcartilage defects in humans and other animals. The subject methods andcompositions may also have prophylactic use in closed as well as openfracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agent is usefulfor repair of craniofacial defects that are congenital, trauma-induced,or caused by oncologic resection, and is also useful in cosmetic plasticsurgery. Further, methods and compositions of the invention may be usedin the treatment of periodontal disease and in other tooth repairprocesses. In certain cases, an ALK4:ActRIIB antagonist (e.g., anALK4:ActRIIB heterodimer), or combinations of such antagonists, mayprovide an environment to attract bone-forming cells, stimulate growthof bone-forming cells, or induce differentiation of progenitors ofbone-forming cells. An ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIBheterodimer), or combinations of such antagonists, of the disclosure mayalso be useful in the treatment of osteoporosis. Further, ALK4:ActRIIBantagonists may be used in repair of cartilage defects andprevention/reversal of osteoarthritis. Examples of heteromeric complexesuseful for inducing bone formation, preventing bone loss, increasingbone mineralization, preventing the demineralization of bone, and/orincreasing bone density as described herein are ALK4:ActRIIBheterodimers.

Rosen et al. (ed) Primer on the Metabolic Bone Diseases and Disorders ofMineral Metabolism, 7^(th) ed. American Society for Bone and MineralResearch, Washington D.C. (incorporated herein by reference) provides anextensive discussion of bone disorders that may be subject to treatmentwith an ALK4:ActRIIB antagonist, or with combinations of suchantagonists. A partial listing is provided herein. Methods andcompositions of the invention can be applied to conditions characterizedby or causing bone loss, such as osteoporosis (including secondaryosteoporosis), hyperparathyroidism, chronic kidney disease mineral bonedisorder, sex hormone deprivation or ablation (e.g. androgen and/orestrogen), glucocorticoid treatment, rheumatoid arthritis, severe burns,hyperparathyroidism, hypercalcemia, hypocalcemia, hypophosphatemia,osteomalacia (including tumor-induced osteomalacia), hyperphosphatemia,vitamin D deficiency, hyperparathyroidism (including familialhyperparathyroidism) and pseudohypoparathyroidism, tumor metastases tobone, bone loss as a consequence of a tumor or chemotherapy, tumors ofthe bone and bone marrow (e.g., multiple myeloma), ischemic bonedisorders, periodontal disease and oral bone loss, Cushing's disease,Paget's disease, thyrotoxicosis, chronic diarrheal state ormalabsorption, renal tubular acidosis, or anorexia nervosa. Methods andcompositions of the invention may also be applied to conditionscharacterized by a failure of bone formation or healing, includingnon-union fractures, fractures that are otherwise slow to heal, fetaland neonatal bone dysplasias (e.g., hypocalcemia, hypercalcemia, calciumreceptor defects and vitamin D deficiency), osteonecrosis (includingosteonecrosis of the jaw) and osteogenesis imperfecta. Additionally, theanabolic effects will cause such antagonists to diminish bone painassociated with bone damage or erosion. As a consequence of theanti-resorptive effects, such antagonists may be useful to treatdisorders of abnormal bone formation, such as osteoblastic tumormetastases (e.g., associated with primary prostate or breast cancer),osteogenic osteosarcoma, osteopetrosis, progressive diaphysealdysplasia, endosteal hyperostosis, osteopoikilosis, and melorheostosis.Other disorders that may be treated include fibrous dysplasia andchondrodysplasias.

In another specific embodiment, the disclosure provides a therapeuticmethod and composition for repairing fractures and other conditionsrelated to cartilage and/or bone defects or periodontal diseases. Theinvention further provides therapeutic methods and compositions forwound healing and tissue repair. The types of wounds include, but arenot limited to, burns, incisions and ulcers. See, e.g., PCT PublicationNo. WO 84/01106. Such compositions comprise a therapeutically effectiveamount of at least one of the ALK4:ActRIIB antagonists of the disclosurein admixture with a pharmaceutically acceptable vehicle, carrier, ormatrix.

In some embodiments, an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIBheterodimer), or combinations of such antagonists, of the disclosure canbe applied to conditions causing bone loss such as osteoporosis,hyperparathyroidism, Cushing's disease, thyrotoxicosis, chronicdiarrheal state or malabsorption, renal tubular acidosis, or anorexianervosa. It is commonly appreciated that being female, having a low bodyweight, and leading a sedentary lifestyle are risk factors forosteoporosis (loss of bone mineral density, leading to fracture risk).However, osteoporosis can also result from the long-term use of certainmedications. Osteoporosis resulting from drugs or another medicalcondition is known as secondary osteoporosis. In Cushing's disease, theexcess amount of cortisol produced by the body results in osteoporosisand fractures. The most common medications associated with secondaryosteoporosis are the corticosteroids, a class of drugs that act likecortisol, a hormone produced naturally by the adrenal glands. Althoughadequate levels of thyroid hormones are needed for the development ofthe skeleton, excess thyroid hormone can decrease bone mass over time.Antacids that contain aluminum can lead to bone loss when taken in highdoses by people with kidney problems, particularly those undergoingdialysis. Other medications that can cause secondary osteoporosisinclude phenytoin (Dilantin) and barbiturates that are used to preventseizures; methotrexate (Rheumatrex, Immunex, Folex PFS), a drug for someforms of arthritis, cancer, and immune disorders; cyclosporine(Sandimmune, Neoral), a drug used to treat some autoimmune diseases andto suppress the immune system in organ transplant patients; luteinizinghormone-releasing hormone agonists (Lupron, Zoladex), used to treatprostate cancer and endometriosis; heparin (Calciparine, Liquaemin), ananticlotting medication; and cholestyramine (Questran) and colestipol(Colestid), used to treat high cholesterol. Bone loss resulting fromcancer therapy is widely recognized and termed cancer therapy-inducedbone loss (CTIBL). Bone metastases can create cavities in the bone thatmay be corrected by treatment with An ALK4:ActRIIB antagonist (e.g., anALK4:ActRIIB heterodimer). Bone loss can also be caused by gum disease,a chronic infection in which bacteria located in gum recesses producetoxins and harmful enzymes.

In a further embodiment, the present disclosure provides methods andtherapeutic agents for treating diseases or disorders associated withabnormal or unwanted bone growth. For example, patients with thecongenital disorder fibrodysplasia ossificans progressiva (FOP) areafflicted by progressive ectopic bone growth in soft tissuesspontaneously or in response to tissue trauma, with a major impact onquality of life. Additionally, abnormal bone growth can occur after hipreplacement surgery and thus ruin the surgical outcome. This is a morecommon example of pathological bone growth and a situation in which thesubject methods and compositions may be therapeutically useful. The samemethods and compositions may also be useful for treating other forms ofabnormal bone growth (e.g., pathological growth of bone followingtrauma, burns or spinal cord injury), and for treating or preventing theundesirable conditions associated with the abnormal bone growth seen inconnection with metastatic prostate cancer or osteosarcoma.

In certain embodiments, an ALK4:ActRIIB antagonist (e.g., anALK4:ActRIIB heterodimer), or combinations of such antagonists, of thedisclosure may be used to promote bone formation in patients withcancer. Patients having certain tumors (e.g. prostate, breast, multiplemyeloma or any tumor causing hyperparathyroidism) are at high risk forbone loss due to tumor-induced bone loss, bone metastases, andtherapeutic agents. Such patients may be treated with a TGF-betasuperfamily heteromultimer complex, or a combination of complexes, evenin the absence of evidence of bone loss or bone metastases. Patients mayalso be monitored for evidence of bone loss or bone metastases, and maybe treated with an ALK4:ActRIIB antagonist in the event that indicatorssuggest an increased risk. Generally, DEXA scans are employed to assesschanges in bone density, while indicators of bone remodeling may be usedto assess the likelihood of bone metastases. Serum markers may bemonitored. Bone specific alkaline phosphatase (B SAP) is an enzyme thatis present in osteoblasts. Blood levels of BSAP are increased inpatients with bone metastasis and other conditions that result inincreased bone remodeling. Osteocalcin and procollagen peptides are alsoassociated with bone formation and bone metastases. Increases in BSAPhave been detected in patients with bone metastasis caused by prostatecancer, and to a lesser degree, in bone metastases from breast cancer.BMP7 levels are high in prostate cancer that has metastasized to bone,but not in bone metastases due to bladder, skin, liver, or lung cancer.Type I carboxy-terminal telopeptide (ICTP) is a crosslink found incollagen that is formed during to the resorption of bone. Since bone isconstantly being broken down and reformed, ICTP will be found throughoutthe body. However, at the site of bone metastasis, the level will besignificantly higher than in an area of normal bone. ICTP has been foundin high levels in bone metastasis due to prostate, lung, and breastcancer. Another collagen crosslink, Type I N-terminal telopeptide (NTx),is produced along with ICTP during bone turnover. The amount of NTx isincreased in bone metastasis caused by many different types of cancerincluding lung, prostate, and breast cancer. Also, the levels of NTxincrease with the progression of the bone metastasis. Therefore, thismarker can be used to both detect metastasis as well as measure theextent of the disease. Other markers of resorption include pyridinolineand deoxypyridinoline. Any increase in resorption markers or markers ofbone metastases indicate the need for therapy with an ALK4:ActRIIBantagonist in a patient.

In another embodiment, an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIBheterodimer), or combinations of such antagonists, may be used inpatients with chronic kidney disease mineral bone disorder (CKD-MBD), abroad syndrome of interrelated skeletal, cardiovascular, andmineral-metabolic disorders arising from kidney disease. CKD-MBDencompasses various skeletal pathologies often referred to as renalosteodystrophy (ROD), which is a preferred embodiment for treatmentwith, an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIB heterodimer), orcombinations of such antagonists. Depending on the relative contributionof different pathogenic factors, ROD is manifested as diverse pathologicpatterns of bone remodeling (Hruska et al., 2008, Chronic kidney diseasemineral bone disorder (CKD-MBD); in Rosen et al. (ed) Primer on theMetabolic Bone Diseases and Disorders of Mineral Metabolism, 7th ed.American Society for Bone and Mineral Research, Washington D.C., pp343-349). At one end of the spectrum is ROD with uremic osteodystrophyand low bone turnover, characterized by a low number of activeremodeling sites, profoundly suppressed bone formation, and low boneresorption. At the other extreme is ROD with hyperparathyroidism, highbone turnover, and osteitis fibrosa. Given that an ALK4:ActRIIBantagonist (e.g., an ALK4:ActRIIB heterodimer), or combinations of suchantagonists, may exert both anabolic and antiresorptive effects, theseagents may be useful in patients across the ROD pathology spectrum.

An ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIB heterodimer), orcombinations of such antagonists, of the disclosure may be conjointlyadministered with other bone-active pharmaceutical agents. Conjointadministration may be accomplished by administration of a singleco-formulation, by simultaneous administration, or by administration atseparate times. ALK4:ActRIIB antagonists may be particularlyadvantageous if administered with other bone-active agents. A patientmay benefit from conjointly receiving an ALK4:ActRIIB antagonist complexand taking calcium supplements, vitamin D, appropriate exercise and/or,in some cases, other medication. Examples of other medications include,bisphosphonates (alendronate, ibandronate and risedronate), calcitonin,estrogens, parathyroid hormone and raloxifene. The bisphosphonates(alendronate, ibandronate and risedronate), calcitonin, estrogens andraloxifene affect the bone remodeling cycle and are classified asanti-resorptive medications. Bone remodeling consists of two distinctstages: bone resorption and bone formation. Anti-resorptive medicationsslow or stop the bone-resorbing portion of the bone-remodeling cycle butdo not slow the bone-forming portion of the cycle. As a result, newformation continues at a greater rate than bone resorption, and bonedensity may increase over time. Teriparatide, a form of parathyroidhormone, increases the rate of bone formation in the bone remodelingcycle. Alendronate is approved for both the prevention (5 mg per day or35 mg once a week) and treatment (10 mg per day or 70 mg once a week) ofpostmenopausal osteoporosis. Alendronate reduces bone loss, increasesbone density and reduces the risk of spine, wrist and hip fractures.Alendronate also is approved for treatment of glucocorticoid-inducedosteoporosis in men and women as a result of long-term use of thesemedications (i.e., prednisone and cortisone) and for the treatment ofosteoporosis in men. Alendronate plus vitamin D is approved for thetreatment of osteoporosis in postmenopausal women (70 mg once a weekplus vitamin D), and for treatment to improve bone mass in men withosteoporosis. Ibandronate is approved for the prevention and treatmentof postmenopausal osteoporosis. Taken as a once-a-month pill (150 mg),ibandronate should be taken on the same day each month. Ibandronatereduces bone loss, increases bone density and reduces the risk of spinefractures. Risedronate is approved for the prevention and treatment ofpostmenopausal osteoporosis. Taken daily (5 mg dose) or weekly (35 mgdose or 35 mg dose with calcium), risedronate slows bone loss, increasesbone density and reduces the risk of spine and non-spine fractures.Risedronate also is approved for use by men and women to prevent and/ortreat glucocorticoid-induced osteoporosis that results from long-termuse of these medications (i.e., prednisone or cortisone). Calcitonin isa naturally occurring hormone involved in calcium regulation and bonemetabolism. In women who are more than 5 years beyond menopause,calcitonin slows bone loss, increases spinal bone density, and mayrelieve the pain associated with bone fractures. Calcitonin reduces therisk of spinal fractures. Calcitonin is available as an injection(50-100 IU daily) or nasal spray (200 IU daily).

A patient may also benefit from conjointly receiving an ALK4:ActRIIBantagonist, or combinations of such antagonists, and additionalbone-active medications. Estrogen therapy (ET)/hormone therapy (HT) isapproved for the prevention of osteoporosis. ET has been shown to reducebone loss, increase bone density in both the spine and hip, and reducethe risk of hip and spinal fractures in postmenopausal women. ET isadministered most commonly in the form of a pill or skin patch thatdelivers a low dose of approximately 0.3 mg daily or a standard dose ofapproximately 0.625 mg daily and is effective even when started afterage 70. When estrogen is taken alone, it can increase a woman's risk ofdeveloping cancer of the uterine lining (endometrial cancer). Toeliminate this risk, healthcare providers prescribe the hormoneprogestin in combination with estrogen (hormone replacement therapy orHT) for those women who have an intact uterus. ET/HT relieves menopausesymptoms and has been shown to have a beneficial effect on bone health.Side effects may include vaginal bleeding, breast tenderness, mooddisturbances and gallbladder disease. Raloxifene, 60 mg a day, isapproved for the prevention and treatment of postmenopausalosteoporosis. It is from a class of drugs called Selective EstrogenReceptor Modulators (SERMs) that have been developed to provide thebeneficial effects of estrogens without their potential disadvantages.Raloxifene increases bone mass and reduces the risk of spine fractures.Data are not yet available to demonstrate that raloxifene can reduce therisk of hip and other non-spine fractures. Teriparatide, a form ofparathyroid hormone, is approved for the treatment of osteoporosis inpostmenopausal women and men who are at high risk for a fracture. Thismedication stimulates new bone formation and significantly increasesbone mineral density. In postmenopausal women, fracture reduction wasnoted in the spine, hip, foot, ribs and wrist. In men, fracturereduction was noted in the spine, but there were insufficient data toevaluate fracture reduction at other sites. Teriparatide isself-administered as a daily injection for up to 24 months.

In other embodiments, an ALK4:ActRIIB antagonist, or combinations ofsuch antagonists, can be used for regulating body fat content in ananimal and for treating or preventing conditions related thereto, andparticularly, health-compromising conditions related thereto. Accordingto the present invention, to regulate (control) body weight can refer toreducing or increasing body weight, reducing or increasing the rate ofweight gain, or increasing or reducing the rate of weight loss, and alsoincludes actively maintaining, or not significantly changing body weight(e.g., against external or internal influences which may otherwiseincrease or decrease body weight). One embodiment of the presentdisclosure relates to regulating body weight by administering to ananimal (e.g., a human) in need thereof a TGF-beta superfamilyheteromultimer complex, or combinations of TGF-beta superfamilyheteromultimer complexes, of the disclosure.

In some embodiments, an ALK4:ActRIIB antagonist, or combinations of suchantagonists, of the present disclosure can be used for reducing bodyweight and/or reducing weight gain in an animal, and more particularly,for treating or ameliorating obesity in patients at risk for orsuffering from obesity. In another specific embodiment, the presentinvention is directed to methods and compounds for treating an animalthat is unable to gain or retain weight (e.g., an animal with a wastingsyndrome). Such methods are effective to increase body weight and/ormass, or to reduce weight and/or mass loss, or to improve conditionsassociated with or caused by undesirably low (e.g., unhealthy) bodyweight and/or mass. In addition, disorders of high cholesterol (e.g.,hypercholesterolemia or dislipidemia) may be treated with anALK4:ActRIIB antagonist, or combinations of such antagonists, of thedisclosure.

In other embodiments, an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIBheterodimer), or combinations of such antagonists, can be used forregulating body fat content in an animal and for treating or preventingconditions related thereto, and particularly, health-compromisingconditions related thereto. According to the present invention, toregulate (control) body weight can refer to reducing or increasing bodyweight, reducing or increasing the rate of weight gain, or increasing orreducing the rate of weight loss, and also includes activelymaintaining, or not significantly changing body weight (e.g., againstexternal or internal influences which may otherwise increase or decreasebody weight). One embodiment of the present disclosure relates toregulating body weight by administering to an animal (e.g., a human) inneed thereof an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIBheterodimer), or combinations of such antagonists. For example, in someembodiments, an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIBheterodimer), or combinations of such antagonists, may be used to treator prevent a disorder or condition selected from obesity (e.g.,abdominal obesity); overweight; insulin resistance; metabolic syndromeand other metabolic diseases or conditions; a lipid disorder such as,low HDL levels, high LDL levels, hyperlipidemia, hypertriglyceridemia ordyslipidemia; lipoprotein aberrations; decreased triglycerides;inflammation (e.g., liver inflammation and/or inflammation of adiposetissue), fatty liver disease; non-alcoholic fatty liver disease;hyperglycemia; impaired glucose tolerance (IGT); hyperinsulinemia; highcholesterol (e.g., high LDL levels and hypercholesterolemia);cardiovascular disease such as, heart disease including coronary heartdisease, congestive heart failure, stroke, peripheral vascular disease,atherosclerosis; arteriosclerosis, and hypertension; Syndrome X;vascular restenosis; neuropathy; retinopathy; neurodegenerative disease;endothelial dysfunction, respiratory dysfunction; pancreatitis;polycystic ovarian syndrome; elevated uric acid levels; haemochromatosis(iron overload); acanthosis nigricans (dark patches on the skin); orcancer (e.g., ovarian, breast, endometrial, and colon cancer); or aanother disorders/conditions associated with one or more of the abovediseases or conditions. In some embodiments, the disease or conditiontreated using an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIBheterodimer), or combinations of such antagonists, is associated withoverweight (e.g., BMI of ≥25 kg/m²), or with too much body fat.

In one embodiment, the disclosure provides a method of reducing bodyweight comprising administering to a subject desiring to reduce bodyweight, or in need thereof, an effective amount of an ALK4:ActRIIBantagonist (e.g., an ALK4:ActRIIB heterodimer), or combinations of suchantagonists. In some embodiments, the subject is overweight (e.g.,pre-obese). In some embodiments, the subject has a body mass index (BMI)of 25 kg/m² or greater. In further embodiments, the subject has a BMI of25 kg/m² to 29.9 kg/m², 30 kg/m² to 39.9 kg/m², 25 kg/m² to 39.9 kg/m²,or 25 kg/m² to 50 kg/m². In some embodiments, the subject is obese. Insome embodiments, the subject has a BMI of 30 kg/m² or greater (e.g., 30to 39.9 kg/m² or 30 kg/m² to 50 kg/m²). In some embodiments, the subjectis morbidly obese. In some embodiments, the subject has a BMI of 40kg/m² or greater. In further embodiments, the subject has a BMI of 40kg/m² to 45 kg/m², or 40 kg/m² to 50 kg/m². In some embodiments, thesubject has central obesity (e.g., excess adiposity in the abdominalregion, including belly fat and/or visceral fat). In some embodiments,the subject has a waist/hip circumference ratio (WHR) of 0.85 orgreater. In some embodiments, the subject has peripheral obesity (e.g.,excess adiposity on the hips). In some embodiments, the subject has type2 diabetes mellitus. The ALK4:ActRIIB antagonist, or combination ofantagonists, may administered alone or as a combination therapy othertype of supportive therapy. For example, in some embodiments, thesupportive therapy is diet and/or exercise.

In one embodiment, the disclosure provides a method of reducing weightgain comprising administering to a subject desiring to reduce weightgain, or in need thereof, an effective amount of an ALK4:ActRIIBantagonist (e.g., an ALK4:ActRIIB heterodimer), or combination of suchantagonists. In some embodiments, the subject is overweight (e.g.,pre-obese). In some embodiments, the subject has a BMI of 25 kg/m² orgreater. In further embodiments, the subject has a BMI of 25 kg/m² to29.9 kg/m², 30 kg/m² to 39.9 kg/m², 25 kg/m² to 39.9 kg/m², or 25 kg/m²to 50 kg/m². In some embodiments, the subject is obese. In someembodiments, the subject has a BMI of 30 kg/m² or greater (e.g., 30 to39.9 kg/m² or 30 kg/m² to 50 kg/m²). In some embodiments, the subject ismorbidly obese. In some embodiments, the subject has a BMI of 40 kg/m²or greater. In further embodiments, the subject has a BMI of 40 kg/m² to45 kg/m², or 40 kg/m² to 50 kg/m². In some embodiments, the subject hastype 2 diabetes mellitus.

Also provided is a method of treating or preventing a disease orcondition associated with excess body weight, comprising administeringto a subject in need of treatment or prevention, an effective amount ofan ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIB heterodimer), orcombination of such antagonists. In one embodiment, the treated orprevented disease or condition is obesity. In one embodiment, thetreated or prevented disease or condition is insulin resistance. In oneembodiment, the treated or prevented disease or condition is a memberselected from the group consisting of: dyslipidemia, hyperlipidemia(total cholesterol level >240 mg/dL), hypercholesterolemia (e.g., totalcholesterol level of >200 mg/dL, >220 mg/dL, >240 mg/dL, >250 mg/dL,or >275 mg/dL), low HDL serum level (e.g., <40 mg/dL, <45 mg/dL, or <50mg/dL), high LDL serum level (e.g., ≥100 mg/dL, ≥130 mg/dL, ≥160 mg/dL,or ≥190 mg/dL), and hypertriglyceridemia (e.g., a fasting TG level of≥150 mg/dL, ≥175 mg/dL, ≥200 mg/dL, ≥300 mg/dL, ≥400 mg/dL, or ≥499mg/dL). In certain instances, the ALK4:ActRIIB antagonists treatment isan adjunct to diet and/or exercise.

In another embodiment the disclosure provides a method of reducing bodyweight in a subject who is overweight. The method includes administeringto an overweight subject an effective amount of an ALK4:ActRIIBantagonist (e.g., an ALK4:ActRIIB heterodimer), or combination of suchantagonists. In some embodiments, the subject has a body mass index(BMI) of 25 kg/m² or greater. In further embodiments, the subject has aBMI of 25 kg/m² to 29.9 kg/m², 30 kg/m² to 39.9 kg/m², 25 kg/m² to 39.9kg/m², or 25 kg/m² to 50 kg/m² or 27 to 40 kg/m². In some embodiments,the subject is obese. In some embodiments, the subject has a BMI of 30kg/m² or greater (e.g., 30 to 39.9 kg/m² or 30 kg/m² to 50 kg/m²). TheALK4:ActRIIB antagonist is administered alone or as a combinationtherapy. In some embodiments, the ALK4:ActRIIB antagonist treatment isan adjunct to diet and/or exercise.

In one embodiment the disclosure provides a method of reducing bodyweight in an obese subject. The method includes administering to thesubject an effective amount of an ALK4:ActRIIB antagonist (e.g., anALK4:ActRIIB heterodimer), or combination of such antagonists. In someembodiments, the subject has a BMI of 30 kg/m² or greater (e.g., 30 to39.9 kg/m² or 30 kg/m² to 50 kg/m². In some embodiments, the subject hasa BMI of 40 kg/m² or greater. In some embodiments, the subject hascentral obesity (e.g., excess adiposity in the abdominal region,including belly fat and/or visceral fat). In some embodiments, thesubject has a waist/hip circumference ratio (WHR) of 0.85 or greater. Insome embodiments, the subject has peripheral obesity (e.g., excessadiposity on the hips). In some embodiments, the ALK4:ActRIIB antagonisttreatment is an adjunct to diet and/or exercise.

In another embodiment, the disclosure provides a method of treatingand/or ameliorating obesity or a disease or condition associated withobesity, comprising administering to an obese subject, an effectiveamount of an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIBheterodimer), or combination of such antagonists. In some embodiments,the subject has a BMI of 30 kg/m² or greater. In further embodiments,the subject has a BMI of 30 to 39.9 kg/m² or 30 kg/m² to 50 kg/m². Insome embodiments, the subject is morbidly obese. In some embodiments,the subject has a body BMI of 40 kg/m² or greater. In furtherembodiments, the subject has a BMI of 40 kg/m² to 45 kg/m², or 40 kg/m²to 50 kg/m²In some embodiments, the subject has type 2 diabetesmellitus. In some embodiments, the subject has a BMI of 30 kg/m² orgreater (e.g., 30 to 39.9 kg/m²). In some embodiments, the subject has aBMI of at least 40 kg/m². In some embodiments, the subject has centralobesity (e.g., excess adiposity in the abdominal region, including bellyfat and/or visceral fat). In some embodiments, the subject has awaist/hip circumference ratio (WHR) of 0.85 or greater. In someembodiments, the subject has peripheral obesity (e.g., excess adiposityon the hips). In some embodiments, the ALK4:ActRIIB antagonist treatmentis an adjunct to diet and/or exercise.

Also provided is a method of treating or preventing a disease orcondition associated with obesity, comprising administering to a subjectin need of treatment or prevention, an effective amount of anALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIB heterodimer), orcombination of such antagonists. In one embodiment, the treated orprevented disease or condition is a member selected from the groupconsisting of: dyslipidemia, hyperlipidemia (total cholesterollevel >240 mg/dL), hypercholesterolemia (e.g., total cholesterol levelof >200 mg/dL, >220 mg/dL, >240 mg/dL, >250 mg/dL, or >275 mg/dL), lowHDL serum level (e.g., <40 mg/dL, <45 mg/dL, or <50 mg/dL), high LDLserum level (e.g., ≥100 mg/dL, ≥130 mg/dL, ≥160 mg/dL, or ≥190 mg/dL),and hypertriglyceridemia (e.g., a fasting TG level of ≥150 mg/dL, ≥175mg/dL, ≥200 mg/dL, ≥300 mg/dL, ≥400 mg/dL, or ≥499 mg/dL). In oneembodiment, the treated or prevented disease or condition iscardiovascular disease. In an additional embodiment, the treated orprevented disease or condition is hypertension (high blood pressure),myocardial infarction, peripheral artery disease, vasoregulatoindysfunction, arteriosclerosis congestive heart failure, atherosclerosis,coronary heart disease, or microvascular disease. In one embodiment, thetreated or prevented disease or condition is liver disease. In oneembodiment, the treated or prevented liver disease or condition isNAFLD. In one embodiment, the liver disease is fatty liver. In oneembodiment, the liver disease is NASH. In another embodiment, thetreated or prevented disease or condition is a member selected from thegroup: steatohepatitis, steatosis, fibrosis, and/or cirrhosis. Incertain instances, the ALK4:ActRIIB antagonist treatment is an adjunctto diet and/or exercise.

In another embodiment, the disclosure provides a method of treating,ameliorating, and/or preventing type 2 diabetes mellitus or a disease orcondition associated with diabetes comprising administering to a subjecthaving type 2 diabetes mellitus, or at risk of developing type 2diabetes, an effective amount of an ALK4:ActRIIB antagonist (e.g., anALK4:ActRIIB heterodimer), or combination of such antagonists. In someembodiments, the subject has a body mass index BMI of 30 kg/m² orgreater (e.g., 30 to 39.9 kg/m²). In some embodiments, the subject has aBMI of at least 40 kg/m². In some embodiments, the subject has centralobesity (e.g., excess adiposity in the abdominal region, including bellyfat and/or visceral fat). In some embodiments, the subject has a WHR of0.85 or greater. In some embodiments, the subject has peripheral obesity(e.g., excess adiposity on the hips). In some embodiments, theALK4:ActRIIB antagonist treatment is an adjunct to diet and/or exercise.

Also provided is a method of treating, ameliorating or preventing adisease or condition associated with diabetes, comprising administeringto a subject having diabetes, an effective amount of an ALK4:ActRIIBantagonist (e.g., an ALK4:ActRIIB heterodimer), or combination of suchantagonists. In one embodiment, the treated or prevented disease orcondition is a member selected from the group consisting of:dyslipidemia, hyperlipidemia (total cholesterol level >240 mg/dL),hypercholesterolemia (e.g., total cholesterol level of >200 mg/dL, >220mg/dL, >240 mg/dL, >250 mg/dL, or >275 mg/dL), low HDL serum level(e.g., <40 mg/dL, <45 mg/dL, or <50 mg/dL), high LDL serum level (e.g.,≥100 mg/dL, ≥130 mg/dL, ≥160 mg/dL, or ≥190 mg/dL), andhypertriglyceridemia (e.g., a fasting TG level of ≥150 mg/dL, ≥175mg/dL, ≥200 mg/dL, ≥300 mg/dL, ≥400 mg/dL, or ≥499 mg/dL). In oneembodiment, the treated or prevented disease or condition iscardiovascular disease. In an additional embodiment, the treated orprevented disease or condition is hypertension (high blood pressure),myocardial infarction, peripheral artery disease, vasoregulatoindysfunction, or arteriosclerosis. In one embodiment, the treated orprevented disease or condition is liver disease. In another embodiment,the treated or prevented disease or condition is a member selected fromthe group: fatty liver disease, steatohepatitis, steatosis, and/orcirrhosis. In one embodiment, the treated or prevented disease orcondition is a member selected from the group consisting of: cataracts,obstructive sleep apnea, phlebitis, gout, osteoarthritis, gallbladderdisease, and high cholesterol. In certain instances, the ALK4:ActRIIBantagonist treatment is an adjunct to diet and/or exercise.

The disclosure also provides a method for improving the blood-lipidprofile in a subject, comprising administering to a subject in need ofsuch treatment an effective amount of an ALK4:ActRIIB antagonist (e.g.,an ALK4:ActRIIB heterodimer), or combination of such antagonists. Insome embodiments, the disclosure provides a method for reducing levelsof LDL cholesterol or increasing levels of HDL-cholesterol. In oneembodiment, the subject has dyslipidemia. In another embodiment, thesubject has elevated serum lipids (e.g., cholesterol(hypercholesterolemia) and/or triglycerides (e.g.,hypertriglyceridemia). In one embodiment the subject has an LDL-C ≥100mg/dL, ≥130 mg/dL, or ≥160 mg/dL). In one embodiment the subject has aTG ≥150 mg/dL, ≥160 mg/dL, ≥170 mg/dL). In one embodiment, the subjecthas elevated plasma insulin levels (hyperinsulinemia; e.g., fastinginsulin level of >20 ug/ml can exceed 100). In some embodiments, thesubject has type II diabetes.

According to one embodiment, the disclosure provides a method oftreating or preventing a metabolic disease or disorder or a conditionassociated with a metabolic disease or disorder, comprisingadministering an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIBheterodimer), or combination of such antagonists, to a subject in needthereof. In one embodiment, the treated metabolic disease, disorder, orcondition is hyperglycemia (e.g., >130 mg/dL in the fasting state orfollowing glucose administration during an oral glucose tolerance test).In one embodiment, the treated metabolic disease, disorder, or conditionis a lipid metabolism disease, disorder, or condition. In oneembodiment, the treated metabolic disease, disorder, or condition isdislipidemia. In a further embodiment, the lipid metabolism disease,disorder, or condition is a member selected from: low HDL levels, highLDL levels, high triglyceride levels, hyperlipidemia, and a lipoproteinaberration. In one embodiment, the subject has a total cholesterol levelof >200 mg/dL, >220 mg/dL, >240 mg/dL, >250 mg/dL, or >275 mg/dL. In oneembodiment, the subject has a HDL serum level of <40 mg/dL, <45 mg/dL,or <50 mg/dL). In one embodiment, the subject has a LDL serum level ≥100mg/dL, ≥130 mg/dL, ≥160 mg/dL, or ≥190 mg/dL. In one embodiment, thesubject has fasting TG level of ≥150 mg/dL, ≥175 mg/dL, ≥200 mg/dL, ≥300mg/dL, ≥400 mg/dL, or ≥499 mg/dL. In one embodiment, the treatedmetabolic disease, disorder, or condition is a glucose metabolismdisease, disorder, or condition. In a further embodiment, the glucosemetabolism disease, disorder, or condition is a member selected from:glucose intolerance, insulin resistance, impaired glucose tolerance(IGT), impaired fasting glucose (IFG). In one embodiment, the treatedmetabolic disease, disorder, or condition is a member selected from thegroup consisting of: high uric acid levels, NAFLD, fatty liver, NASH,and polycystic ovarian syndrome. In one embodiment, the treated subjecthas hyperinsulinemia. In one embodiment, the treated subject is obese(e.g., the subject has abdominal obesity). In another embodiment, thetreated subject has type II diabetes.

Metabolic syndrome is a condition involving a set of disorders thatenhances the risk of heart disease. The major components of metabolicsyndrome are excess weight, the cardiovascular parameters (high bloodpressure, dyslipidemia, high levels of triglycerides and/or low levelsof HDL in the blood), atherosclerosis, diabetes, and/or insulinresistance. A subject having several of these components, i.e. metabolicsyndrome, is highly prone to heart disease, though each component is arisk factor. The disclosure also provides a method for treating orpreventing 1, 2, 3, or more of the above components of metabolicsyndrome, comprising administering to a subject in need of treatment aneffective amount of an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIBheterodimer), or combination of such antagonists.

Additionally provided is a method of treating, preventing orameliorating a cardiovascular disease or condition, comprisingadministering an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIBheterodimer), or combination of such antagonists, to a subject in needthereof. In one embodiment, the treated, prevented, or amelioratedcardiovascular disease or condition is atherosclerosis. In oneembodiment, the treated, prevented, or ameliorated cardiovasculardisease or condition is hypertension (e.g., blood pressure >130/80 mmHgor >140/90 mmHg, in a resting state. In one embodiment, thecardiovascular disease is atherosclerosis (coronary heart diseasedisease).

In one embodiment, the disclosure provides a method for treating and/orameliorating an inflammatory liver disease or condition that comprisesadministering an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIBheterodimer), or combination of such antagonists, to a subject in needthereof. In one embodiment, the disease or condition is NAFLD. In afurther embodiment, the disease or condition is fatty liver. In afurther embodiment, the disease or condition is steatosis (e.g.,nonalcoholic steatohepatitis (NASH)). In a further embodiment, thedisease or condition is alcoholic fatty liver disease.

This disclosure also provides a method of improving glycemic control,comprising administering to a subject in need of treatment an effectiveamount of an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIBheterodimer). In one embodiment, the subject is administered has afasting blood sugar level of >130, >135, >140, >145, or >150 mg/dL. Inone embodiment, the subject is administered has a postprandial bloodsugar level of >180, >185, >190, >195, or >200 mg/dL 2 hours aftereating. In certain instances, the ALK4:ActRIIB antagonist treatment isan adjunct to diet and/or exercise. The administration can also reducebody weight or treat obesity. In certain instances, the subject has type2 diabetes mellitus. In certain instances, the subject has a BMI of 27to 40 kg/m2. In certain instances, the subject has a BMI of 30 to 39.9kg/m2. In certain instances, the subject has a BMI of at least 40. Incertain instances, the subject is overweight. In certain instances, thesubject is obese. An improvement in glycemic control can be assessedusing techniques known in the art such as a mixed-meal test.

The disclosure also provides compositions and methods for treating,preventing or ameliorating hyperglycemia or a condition associated withhyperglycemia in a subject comprising administering to a subject in needof such treatment an effective amount of an ALK4:ActRIIB antagonist(e.g., an ALK4:ActRIIB heterodimer). In one embodiment, the subject isadministered has a fasting blood sugar level of >130, >135, >140, >145,or >150 mg/dL. In one embodiment, the subject is administered has apostprandial blood sugar level of >180, >185, >190, >195, or >200 mg/dL2 hours after eating. In one embodiment, the result of the treatment,prevention or amelioration is a member selected from the groupconsisting of: a decrease in serum levels of glucose, a decrease inserum levels of triglycerides, a decrease in serum levels of insulin,and/or a decrease in serum levels of non-esterified fatty acids, ascompared to serum levels in the subject prior to treatment. In oneembodiment, the result of the treatment, prevention or amelioration isan increase in body temperature of about 0.4° C. to 1° C. as compared tobody temperature of the subject prior to treatment. In some embodiments,the ALK4:ActRIIB treatment also reduces body weight of the subject.

In another embodiment, the disclosure provides a method of decreasingplasma insulin levels in a subject, comprising administering aneffective amount of an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIBheterodimer), to a subject in need of such treatment. In one embodiment,the subject has a fasting blood sugar level of >130, >135, >140, >145,or >150 mg/dL. In one embodiment, the subject has a postprandial bloodsugar level of >180, >185, >190, >195, or >200 mg/dL 2 hours aftereating. In one embodiment, the subject is overweight. In one embodiment,the subject is obese. In another embodiment, the subject has type 2diabetes.

The disclosure also provides compositions and methods for treating,preventing or ameliorating hyperglycemia or a condition associated withhyperglycemia in a subject comprising administering to a subject in needof such treatment an effective amount of an ALK4:ActRIIB antagonist(e.g., an ALK4:ActRIIB heterodimer). In one embodiment, the subject hasa fasting blood sugar level of >130, >135, >140, >145, or >150 mg/dL. Inone embodiment, the subject has a postprandial blood sugar levelof >180, >185, >190, >195, or >200 mg/dL 2 hours after eating. In oneembodiment, the result of the treatment, prevention or amelioration is amember selected from the group consisting of: a decrease in serum levelsof glucose, a decrease in serum levels of triglycerides, a decrease inserum levels of insulin, and/or a decrease in serum levels ofnon-esterified fatty acids, as compared to serum levels in the subjectprior to treatment. In one embodiment, the result of the treatment,prevention or amelioration is an increase in body temperature of about0.4° C. to 1° C. as compared to body temperature of the subject prior totreatment. In some embodiments, the ALK4:ActRIIB antagonist treatmentalso reduces body weight of the subject.

In another embodiment, the disclosure provides a method of decreasingplasma insulin levels in a subject, comprising administering aneffective amount of an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIBheterodimer), or combination of such antagonists, to a subject in needof such treatment. In one embodiment, the subject has a fasting bloodsugar level of >130, >135, >140, >145, or >150 mg/dL. In one embodiment,the has a postprandial blood sugar level of >180, >185, >190, >195,or >200 mg/dL 2 hours after eating. In one embodiment, the subject isoverweight. In one embodiment, the subject is obese. In anotherembodiment, the subject has type 2 diabetes.

In another embodiment, the disclosure provides a method of treating,preventing, or ameliorating liver disease in a subject, comprisingadministering an effective amount of an ALK4:ActRIIB antagonist (e.g.,an ALK4:ActRIIB heterodimer), or combination of such antagonists, to asubject having a liver disease. In one embodiment, the subject hasinflammation of the liver. In one embodiment, the subject has NAFLD. Inon embodiment the subject has fatty liver. In another embodiment, thesubject has NASH. In on embodiment the subject has fatty liver. Inanother embodiment, the subject has alcoholic fatty liver disease. Inone embodiment, the treated, prevented or ameliorated liver disease isfibrosis, scarring, cirrhosis, or liver failure. In another embodiment,the treated, prevented or ameliorated liver disease is liver cancer. Inone embodiment, the subject is overweight. In another embodiment, thesubject is obese. In another embodiment, the subject has type 2diabetes.

Fibrosis generally refers to an excessive deposition of both collagenfibers and extracellular matrix combined with a relative decrease ofcell number in an organ or tissue. While this process is an importantfeature of natural wound healing following injury, fibrosis can lead topathological damage in various tissue and organs including, for example,the lungs, kidneys, liver, bone, muscle, and skin. The role TGF-beta infibrosis has been extensively study. However, other TGF-beta superfamilyligands have also been implicated in fibrosis including, for example,activins (e.g., activin A and activin B) and GDF8 [Hedger et al (2013)Cytokine and Growth Factor Reviews 24:285-295; Hardy et al. (2015) 93:567-574; and Cantini et al. (2008) J Sex Med 5:1607-1622]. Therefore, insome embodiments, an ALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIBheterodimer), or combinations of such antagonists, of the presentdisclosure can be used to treat fibrosis, particularlyfibrosis-associated disorders and conditions. For example, anALK4:ActRIIB antagonist (e.g., an ALK4:ActRIIB heterodimer), orcombinations of such antagonists, may be used to treat or prevent one ormore of: pulmonary fibrosis, hypersensitivity pneumonitis, idiopathicfibrosis, tuberculosis, pneumonia, cystic fibrosis, asthma, chronicobstructive pulmonary disease (COPD), emphysema, renal (kidney)fibrosis, renal (kidney) failure, chronic renal (kidney) disease, bonefibrosis, myelofibrosis, rheumatoid arthritis, systemic lupuserythematosus, scleroderma, sarcoidosis, granulomatosis withpolyangiitis, Peyronie's disease, liver fibrosis, Wilson's disease,glycogen storage diseases (particularly types III, IV, IX, and X),iron-overload, Gaucher disease, Zellweger syndrome, nonalcoholic andalcoholic steatohepatitis, biliary cirrhosis, sclerosing cholangitis,Budd-Chiari syndrome, surgery-associated fibrosis, Crohn's disease,Duputren's contracture, mediastinal fibrosis, nephrogeneic fibrosis,retroperitoneal fibrosis, atrial fibrosis, endomyocardial fibrosis,pancreatic fibrosis.

The kidneys maintain many features of the blood, including volume, pHbalance, electrolyte concentrations, and blood pressure, as well asbearing responsibility for toxin and waste filtration. These functionsdepend upon the intricate structure of the kidney nephrons, constantflow of blood through the various capillaries of the kidney, and theregulation of the kidney by signals from the rest of the body, includingendocrine hormones. Problems with kidney function manifest by directmechanisms (e.g. genetic defects, infection, or toxin exposure) and byindirect mechanisms progressively proceeding from long term stressorslike hypertrophy and hyperfiltration (themselves often a result of moredirect insults to kidney function). Due to the central role of thekidney in blood maintenance and waste secretion, kidney-associateddisease manifestations are many and varied; they can be reviewed inHarrison's Principles of Internal Medicine, 18^(th) edition, McGrawHill, N.Y., Part 13, Chp 277-289.

As described herein, an ALK4:ActRIIB antagonist had various beneficialeffects in a kidney disease model. In particular, treatment with anALK4:ActRIIB heteromultimer reduced kidney tissue damage, inflammation,and fibrosis in subjects having unilateral ureteral obstruction. Thesedata indicate that ALK4:ActRIIB antagonists may be used to treat orprevent kidney disease, particularly treating or preventing variouscomplications (manifestations) of kidney disease including, for example,kidney tissue damage, inflammation, and/or fibrosis.

Therefore, methods of this invention can be applied to variouskidney-associated diseases or conditions. As used herein,“kidney-associated disease or condition” can refer to any disease,disorder, or condition that affects the kidneys or the renal system.Examples of kidney-associated diseases or conditions include, but arenot limited to, chronic kidney diseases (or failure), acute kidneydiseases (or failure), primary kidney diseases, non-diabetic kidneydiseases, glomerulonephritis, interstitial nephritis, diabetic kidneydiseases, diabetic nephropathy, glomerulosclerosis, rapid progressiveglomerulonephritis, renal fibrosis, Alport syndrome, IDDM nephritis,mesangial proliferative glomerulonephritis, membranoproliferativeglomerulonephritis, crescentic glomerulonephritis, renal interstitialfibrosis, focal segmental glomerulosclerosis, membranous nephropathy,minimal change disease, pauci-immune rapid progressiveglomerulonephritis, IgA nephropathy, polycystic kidney disease, Dent'sdisease, nephrocytinosis, Heymann nephritis, autosomal dominant (adult)polycystic kidney disease, autosomal recessive (childhood) polycystickidney disease, acute kidney injury, nephrotic syndrome, renal ischemia,podocyte diseases or disorders, proteinuria, glomerular diseases,membranous glomerulonephritis, focal segmental glomerulonephritis,pre-eclampsia, eclampsia, kidney lesions, collagen vascular diseases,benign orthostatic (postural) proteinuria, IgM nephropathy, membranousnephropathy, sarcoidosis, diabetes mellitus, kidney damage due to drugs,Fabry's disease, aminoaciduria, Fanconi syndrome, hypertensivenephrosclerosis, interstitial nephritis, Sickle cell disease,hemoglobinuria, myoglobinuria, Wegener's Granulomatosis, GlycogenStorage Disease Type 1, chronic kidney disease, chronic renal failure,low Glomerular Filtration Rate (GFR), nephroangiosclerosis, lupusnephritis, ANCA-positive pauci-immune crescentic glomerulonephritis,chronic allograft nephropathy, nephrotoxicity, renal toxicity, kidneynecrosis, kidney damage, glomerular and tubular injury, kidneydysfunction, nephritic syndrome, acute renal failure, chronic renalfailure, proximal tubal dysfunction, acute kidney transplant rejection,chronic kidney transplant rejection, non-IgA mesangioproliferativeglomerulonephritis, postinfectious glomerulonephritis, vasculitides withrenal involvement of any kind, any hereditary renal disease, anyinterstitial nephritis, renal transplant failure, kidney cancer, kidneydisease associated with other conditions (e.g., hypertension, diabetes,and autoimmune disease), Dent's disease, nephrocytinosis, Heymannnephritis, a primary kidney disease, a collapsing glomerulopathy, adense deposit disease, a cryoglobulinemia-associated glomerulonephritis,an Henoch-Schonlein disease, a postinfectious glomerulonephritis, abacterial endocarditis, a microscopic polyangitis, a Churg-Strausssyndrome, an anti-GBM-antibody mediated glomerulonephritis, amyloidosis,a monoclonal immunoglobulin deposition disease, a fibrillaryglomerulonephritis, an immunotactoid glomerulopathy, ischemic tubularinjury, a medication-induced tubulo-interstitial nephritis, a toxictubulo-interstitial nephritis, an infectious tubulo-interstitialnephritis, a bacterial pyelonephritis, a viral infectioustubulo-interstitial nephritis which results from a polyomavirusinfection or an HIV infection, a metabolic-induced tubulo-interstitialdisease, a mixed connective disease, a cast nephropathy, a crystalnephropathy which may results from urate or oxalate or drug-inducedcrystal deposition, an acute cellular tubulo-interstitial allograftrejection, a tumoral infiltrative disease which results from a lymphomaor a post-transplant lymphoproliferative disease, an obstructive diseaseof the kidney, vascular disease, a thrombotic microangiopathy, anephroangiosclerosis, an atheroembolic disease, a mixed connectivetissue disease, a polyarteritis nodosa, a calcineurin-inhibitorinduced-vascular disease, an acute cellular vascular allograftrejection, an acute humoral allograft rejection, early renal functiondecline (ERFD), end stage renal disease (ESRD), renal vein thrombosis,acute tubular necrosis, acute interstitial nephritis, establishedchronic kidney disease, renal artery stenosis, ischemic nephropathy,uremia, drug and toxin-induced chronic tubulointerstitial nephritis,reflux nephropathy, kidney stones, Goodpasture's syndrome, normocyticnormochromic anemia, renal anemia, diabetic chronic kidney disease,IgG4-related disease, von Hippel-Lindau syndrome, tuberous sclerosis,nephronophthisis, medullary cystic kidney disease, renal cell carcinoma,adenocarcinoma, nephroblastoma, lymphoma, leukemia, hyposialylationdisorder, chronic cyclosporine nephropathy, renal reperfusion injury,renal dysplasia, azotemia, bilateral arterial occlusion, acute uric acidnephropathy, hypovolemia, acute bilateral obstructive uropathy,hypercalcemic nephropathy, hemolytic uremic syndrome, acute urinaryretention, malignant nephrosclerosis, postpartum glomerulosclerosis,scleroderma, non-Goodpasture's anti-GBM disease, microscopicpolyarteritis nodosa, allergic granulomatosis, acute radiationnephritis, post-streptococcal glomerulonephritis, Waldenstrom'smacroglobulinemia, analgesic nephropathy, arteriovenous fistula,arteriovenous graft, dialysis, ectopic kidney, medullary sponge kidney,renal osteodystrophy, solitary kidney, hydronephrosis, microalbuminuria,uremia, haematuria, hyperlipidemia, hypoalbuminaemia, lipiduria,acidosis, hyperkalemia, and edema.

In some embodiments, an ALK4:ActRIIB antagonist, or combinations of suchantagonists, of the present disclosure (e.g., ALK4:ActRIIBheteromultimers such as an ALK4:ActRIIB heterodimer) may be used totreat or prevent chronic kidney disease, optionally in combination withone or more supportive therapies for treating chronic kidney disease. Insome embodiments, an ALK4:ActRIIB antagonist, or combinations of suchantagonists, of the present disclosure (e.g., ALK4:ActRIIBheteromultimers such as an ALK4:ActRIIB heterodimer) may be used totreat or prevent one or more complications (symptoms or manifestations)of chronic kidney disease, optionally in combination with one or moresupportive therapies for treating chronic kidney disease. In someembodiments, an ALK4:ActRIIB antagonist, or combinations of suchantagonists, of the present disclosure (e.g., ALK4:ActRIIBheteromultimers such as an ALK4:ActRIIB heterodimer) may be used totreat or prevent end-stage kidney failure, optionally in combinationwith one or more supportive therapies for treating end-stage kidneydisease. Chronic kidney disease (CKD), also known as chronic renaldisease, is a progressive loss in renal function over a period of monthsor years. The symptoms of worsening kidney function may include feelinggenerally unwell and experiencing a reduced appetite. Often, chronickidney disease is diagnosed as a result of screening of people known tobe at risk of kidney problems, such as those with high blood pressure ordiabetes and those with a blood relative with CKD. This disease may alsobe identified when it leads to one of its recognized complications, suchas cardiovascular disease, anemia, or pericarditis. Recent professionalguidelines classify the severity of CKD in five stages, with stage 1being the mildest and usually causing few symptoms and stage 5 being asevere illness with poor life expectancy if untreated. Stage 5 CKD isoften called end-stage kidney disease, end-stage renal disease, orend-stage kidney failure, and is largely synonymous with the nowoutdated terms chronic renal failure or chronic kidney failure; andusually means the patient requires renal replacement therapy, which mayinvolve a form of dialysis, but ideally constitutes a kidney transplant.CKD is initially without specific symptoms and is generally onlydetected as an increase in serum creatinine or protein in the urine. Asthe kidney function decreases and various symptoms may manifest asdescribed below. Blood pressure may be increased due to fluid overloadand production of vasoactive hormones created by the kidney via therenin-angiotensin system, increasing one's risk of developinghypertension and/or suffering from congestive heart failure. Urea mayaccumulate, leading to azotemia and ultimately uremia (symptoms rangingfrom lethargy to pericarditis and encephalopathy). Due to its highsystemic circulation, urea is excreted in eccrine sweat at highconcentrations and crystallizes on skin as the sweat evaporates (“uremicfrost”). Potassium may accumulate in the blood (hyperkalemia with arange of symptoms including malaise and potentially fatal cardiacarrhythmias). Hyperkalemia usually does not develop until the glomerularfiltration rate falls to less than 20-25 ml/min/1.73 m2, at which pointthe kidneys have decreased ability to excrete potassium. Hyperkalemia inCKD can be exacerbated by acidemia (which leads to extracellular shiftof potassium) and from lack of insulin. Erythropoietin synthesis may bedecreased causing anemia. Fluid volume overload symptoms may occur,ranging from mild edema to life-threatening pulmonary edema.Hyperphosphatemia, due to reduced phosphate excretion, may occurgenerally following the decrease in glomerular filtration.Hyperphosphatemia is associated with increased cardiovascular risk,being a direct stimulus to vascular calcification. Hypocalcemia maymanifest, which is generally caused by stimulation of fibroblast growthfactor-23. Osteocytes are responsible for the increased production ofFGF23, which is a potent inhibitor of the enzyme 1-alpha-hydroxylase(responsible for the conversion of 25-hydroxycholecalciferol into 1,25dihydroxyvitamin D3). Later, this progresses to secondaryhyperparathyroidism, renal osteodystrophy, and vascular calcificationthat further impairs cardiac function. Metabolic acidosis (due toaccumulation of sulfates, phosphates, uric acid etc.) may occur andcause altered enzyme activity by excess acid acting on enzymes; and alsoincreased excitability of cardiac and neuronal membranes by thepromotion of hyperkalemia due to excess acid (acidemia). Acidosis isalso due to decreased capacity to generate enough ammonia from the cellsof the proximal tubule. Iron deficiency anemia, which increases inprevalence as kidney function decreases, is especially prevalent inthose requiring haemodialysis. It is multifactoral in cause, butincludes increased inflammation, reduction in erythropoietin, andhyperuricemia leading to bone marrow suppression. People with CKD sufferfrom accelerated atherosclerosis and are more likely to developcardiovascular disease than the general population. Patients afflictedwith CKD and cardiovascular disease tend to have significantly worseprognoses than those suffering only from the latter.

As used herein, “in combination with”, “combinations of”, or “conjointadministration” refers to any form of administration such thatadditional therapies (e.g., second, third, fourth, etc.) are stilleffective in the body (e.g., multiple compounds are simultaneouslyeffective in the patient, which may include synergistic effects of thosecompounds). Effectiveness may not correlate to measurable concentrationof the agent in blood, serum, or plasma. For example, the differenttherapeutic compounds can be administered either in the same formulationor in separate formulations, either concomitantly or sequentially, andon different schedules. Thus, an individual who receives such treatmentcan benefit from a combined effect of different therapies. One or moreALK4:ActRIIB antagonists of the disclosure can be administeredconcurrently with, prior to, or subsequent to, one or more otheradditional agents or supportive therapies. In general, each therapeuticagent will be administered at a dose and/or on a time scheduledetermined for that particular agent. The particular combination toemploy in a regimen will take into account compatibility of theantagonist of the present disclosure with the therapy and/or thedesired.

7. Pharmaceutical Compositions

In certain aspects, ALK4:ActRIIB antagonists (e.g., ALK4:ActRIIBheteromultimers), or combinations of such antagonists, of the presentdisclosure can be administered alone or as a component of apharmaceutical formulation (also referred to as a therapeuticcomposition or pharmaceutical composition). A pharmaceutical formationrefers to a preparation which is in such form as to permit thebiological activity of an active ingredient (e.g., an agent of thepresent disclosure) contained therein to be effective and which containsno additional components which are unacceptably toxic to a subject towhich the formulation would be administered. The subject compounds maybe formulated for administration in any convenient way for use in humanor veterinary medicine. For example, one or more agents of the presentdisclosure may be formulated with a pharmaceutically acceptable carrier.A pharmaceutically acceptable carrier refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isgenerally nontoxic to a subject. A pharmaceutically acceptable carrierincludes, but is not limited to, a buffer, excipient, stabilizer, and/orpreservative. In general, pharmaceutical formulations for use in thepresent disclosure are in a pyrogen-free, physiologically-acceptableform when administered to a subject. Therapeutically useful agents otherthan those described herein, which may optionally be included in theformulation as described above, may be administered in combination withthe subject agents in the methods of the present disclosure.

In certain embodiments, compositions will be administered parenterally[e.g., by intravenous (I. V.) injection, intraarterial injection,intraosseous injection, intramuscular injection, intrathecal injection,subcutaneous injection, or intradermal injection]. Pharmaceuticalcompositions suitable for parenteral administration may comprise one ormore agents of the disclosure in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use. Injectable solutions or dispersions maycontain antioxidants, buffers, bacteriostats, suspending agents,thickening agents, or solutes which render the formulation isotonic withthe blood of the intended recipient. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalformulations of the present disclosure include water, ethanol, polyols(e.g., glycerol, propylene glycol, polyethylene glycol, etc.), vegetableoils (e.g., olive oil), injectable organic esters (e.g., ethyl oleate),and suitable mixtures thereof. Proper fluidity can be maintained, forexample, by the use of coating materials (e.g., lecithin), by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

In some embodiments, a therapeutic method of the present disclosureincludes administering the pharmaceutical composition systemically, orlocally, from an implant or device. Further, the pharmaceuticalcomposition may be encapsulated or injected in a form for delivery to atarget tissue site (e.g., bone marrow or muscle). In certainembodiments, compositions of the present disclosure may include a matrixcapable of delivering one or more of the agents of the presentdisclosure to a target tissue site (e.g., bone marrow or muscle),providing a structure for the developing tissue and optimally capable ofbeing resorbed into the body. For example, the matrix may provide slowrelease of one or more agents of the present disclosure. Such matricesmay be formed of materials presently in use for other implanted medicalapplications.

The choice of matrix material may be based on one or more of:biocompatibility, biodegradability, mechanical properties, cosmeticappearance, and interface properties. The particular application of thesubject compositions will define the appropriate formulation. Potentialmatrices for the compositions may be biodegradable and chemicallydefined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylacticacid, and polyanhydrides. Other potential materials are biodegradableand biologically well-defined including, for example, bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenon-biodegradable and chemically defined including, for example,sintered hydroxyapatite, bioglass, aluminates, or other ceramics.Matrices may be comprised of combinations of any of the above mentionedtypes of material including, for example, polylactic acid andhydroxyapatite or collagen and tricalciumphosphate. The bioceramics maybe altered in composition (e.g., calcium-aluminate-phosphate) andprocessing to alter one or more of pore size, particle size, particleshape, and biodegradability.

In certain embodiments, pharmaceutical compositions of presentdisclosure can be administered topically. “Topical application” or“topically” means contact of the pharmaceutical composition with bodysurfaces including, for example, the skin, wound sites, and mucousmembranes. The topical pharmaceutical compositions can have variousapplication forms and typically comprises a drug-containing layer, whichis adapted to be placed near to or in direct contact with the tissueupon topically administering the composition. Pharmaceuticalcompositions suitable for topical administration may comprise one ormore one or more ALK4:ActRIIB antagonists of the disclosure incombination formulated as a liquid, a gel, a cream, a lotion, anointment, a foam, a paste, a putty, a semi-solid, or a solid.Compositions in the liquid, gel, cream, lotion, ointment, foam, paste,or putty form can be applied by spreading, spraying, smearing, dabbingor rolling the composition on the target tissue. The compositions alsomay be impregnated into sterile dressings, transdermal patches,plasters, and bandages. Compositions of the putty, semi-solid or solidforms may be deformable. They may be elastic or non-elastic (e.g.,flexible or rigid). In certain aspects, the composition forms part of acomposite and can include fibers, particulates, or multiple layers withthe same or different compositions.

Topical compositions in the liquid form may include pharmaceuticallyacceptable solutions, emulsions, microemulsions, and suspensions. Inaddition to the active ingredient(s), the liquid dosage form may containan inert diluent commonly used in the art including, for example, wateror other solvent, a solubilizing agent and/or emulsifier [e.g., ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, or 1,3-butylene glycol, anoil (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesameoil), glycerol, tetrahydrofuryl alcohol, a polyethylene glycol, a fattyacid ester of sorbitan, and mixtures thereof].

Topical gel, cream, lotion, ointment, semi-solid or solid compositionsmay include one or more thickening agents, such as a polysaccharide,synthetic polymer or protein-based polymer. In one embodiment of theinvention, the gelling agent herein is one that is suitably nontoxic andgives the desired viscosity. The thickening agents may include polymers,copolymers, and monomers of: vinylpyrrolidones, methacrylamides,acrylamides N-vinylimidazoles, carboxy vinyls, vinyl esters, vinylethers, silicones, polyethyleneoxides, polyethyleneglycols,vinylalcohols, sodium acrylates, acrylates, maleic acids,NN-dimethylacrylamides, diacetone acrylamides, acrylamides, acryloylmorpholine, pluronic, collagens, polyacrylamides, polyacrylates,polyvinyl alcohols, polyvinylenes, polyvinyl silicates, polyacrylatessubstituted with a sugar (e.g., sucrose, glucose, glucosamines,galactose, trehalose, mannose, or lactose), acylamidopropane sulfonicacids, tetramethoxyorthosilicates, methyltrimethoxyorthosilicates,tetraalkoxyorthosilicates, trialkoxyorthosilicates, glycols, propyleneglycol, glycerine, polysaccharides, alginates, dextrans, cyclodextrin,celluloses, modified celluloses, oxidized celluloses, chitosans,chitins, guars, carrageenans, hyaluronic acids, inulin, starches,modified starches, agarose, methylcelluloses, plant gums, hylaronans,hydrogels, gelatins, glycosaminoglycans, carboxymethyl celluloses,hydroxyethyl celluloses, hydroxy propyl methyl celluloses, pectins,low-methoxy pectins, cross-linked dextrans, starch-acrylonitrile graftcopolymers, starch sodium polyacrylate, hydroxyethyl methacrylates,hydroxyl ethyl acrylates, polyvinylene, polyethylvinylethers, polymethylmethacrylates, polystyrenes, polyurethanes, polyalkanoates, polylacticacids, polylactates, poly(3-hydroxybutyrate), sulfonated hydrogels, AMPS(2-acrylamido-2-methyl-1-propanesulfonic acid), SEM(sulfoethylmethacrylate), SPM (sulfopropyl methacrylate), SPA(sulfopropyl acrylate),N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine,methacryllic acid amidopropyl-dimethyl ammonium sulfobetaine, SPI(itaconic acid-bis(1-propyl sulfonizacid-3) ester di-potassium salt),itaconic acids, AMBC (3-acrylamido-3-methylbutanoic acid),beta-carboxyethyl acrylate (acrylic acid dimers), and maleicanhydride-methylvinyl ether polymers, derivatives thereof, saltsthereof, acids thereof, and combinations thereof. In certainembodiments, pharmaceutical compositions of present disclosure can beadministered orally, for example, in the form of capsules, cachets,pills, tablets, lozenges (using a flavored basis such as sucrose andacacia or tragacanth), powders, granules, a solution or a suspension inan aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquidemulsion, or an elixir or syrup, or pastille (using an inert base, suchas gelatin and glycerin, or sucrose and acacia), and/or a mouth wash,each containing a predetermined amount of a compound of the presentdisclosure and optionally one or more other active ingredients. Acompound of the present disclosure and optionally one or more otheractive ingredients may also be administered as a bolus, electuary, orpaste.

In solid dosage forms for oral administration (e.g., capsules, tablets,pills, dragees, powders, and granules), one or more compounds of thepresent disclosure may be mixed with one or more pharmaceuticallyacceptable carriers including, for example, sodium citrate, dicalciumphosphate, a filler or extender (e.g., a starch, lactose, sucrose,glucose, mannitol, and silicic acid), a binder (e.g.carboxymethylcellulose, an alginate, gelatin, polyvinyl pyrrolidone,sucrose, and acacia), a humectant (e.g., glycerol), a disintegratingagent (e.g., agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, a silicate, and sodium carbonate), a solution retardingagent (e.g. paraffin), an absorption accelerator (e.g. a quaternaryammonium compound), a wetting agent (e.g., cetyl alcohol and glycerolmonostearate), an absorbent (e.g., kaolin and bentonite clay), alubricant (e.g., a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate), a coloring agent, andmixtures thereof. In the case of capsules, tablets, and pills, thepharmaceutical formulation (composition) may also comprise a bufferingagent. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using one or moreexcipients including, e.g., lactose or a milk sugar as well as a highmolecular-weight polyethylene glycol.

Liquid dosage forms for oral administration of the pharmaceuticalcomposition may include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and elixirs. In additionto the active ingredient(s), the liquid dosage form may contain an inertdiluent commonly used in the art including, for example, water or othersolvent, a solubilizing agent and/or emulsifier [e.g., ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, or 1,3-butylene glycol, an oil (e.g.,cottonseed, groundnut, corn, germ, olive, castor, and sesame oil),glycerol, tetrahydrofuryl alcohol, a polyethylene glycol, a fatty acidester of sorbitan, and mixtures thereof]. Besides inert diluents, theoral formulation can also include an adjuvant including, for example, awetting agent, an emulsifying and suspending agent, a sweetening agent,a flavoring agent, a coloring agent, a perfuming agent, a preservativeagent, and combinations thereof.

Suspensions, in addition to the active compounds, may contain suspendingagents including, for example, an ethoxylated isostearyl alcohol,polyoxyethylene sorbitol, a sorbitan ester, microcrystalline cellulose,aluminum metahydroxide, bentonite, agar-agar, tragacanth, andcombinations thereof.

Prevention of the action and/or growth of microorganisms may be ensuredby the inclusion of various antibacterial and antifungal agentsincluding, for example, paraben, chlorobutanol, and phenol sorbic acid.

In certain embodiments, it may be desirable to include an isotonic agentincluding, for example, a sugar or sodium chloride into thecompositions. In addition, prolonged absorption of an injectablepharmaceutical form may be brought about by the inclusion of an agentthat delay absorption including, for example, aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the one or more of the agents of the present disclosure. In the caseof a ALK4:ActRIIB antagonist that promotes red blood cell formation,various factors may include, but are not limited to, the patient's redblood cell count, hemoglobin level, the desired target red blood cellcount, the patient's age, the patient's sex, the patient's diet, theseverity of any disease that may be contributing to a depressed redblood cell level, the time of administration, and other clinicalfactors. The addition of other known active agents to the finalcomposition may also affect the dosage. Progress can be monitored byperiodic assessment of one or more of red blood cell levels, hemoglobinlevels, reticulocyte levels, and other indicators of the hematopoieticprocess.

In certain embodiments, the present disclosure also provides genetherapy for the in vivo production of one or more of the agents of thepresent disclosure. Such therapy would achieve its therapeutic effect byintroduction of the agent sequences into cells or tissues having one ormore of the disorders as listed above. Delivery of the agent sequencescan be achieved, for example, by using a recombinant expression vectorsuch as a chimeric virus or a colloidal dispersion system. Preferredtherapeutic delivery of one or more of agent sequences of the disclosureis the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or an RNA virus(e.g., a retrovirus). The retroviral vector may be a derivative of amurine or avian retrovirus. Examples of retroviral vectors in which asingle foreign gene can be inserted include, but are not limited to:Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus(RSV). A number of additional retroviral vectors can incorporatemultiple genes. All of these vectors can transfer or incorporate a genefor a selectable marker so that transduced cells can be identified andgenerated. Retroviral vectors can be made target-specific by attaching,for example, a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using an antibody. Those of skill in the art willrecognize that specific polynucleotide sequences can be inserted intothe retroviral genome or attached to a viral envelope to allow targetspecific delivery of the retroviral vector containing one or more of theagents of the present disclosure.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes (gag, pol, and env),by conventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for one or more of the agents of thepresent disclosure is a colloidal dispersion system. Colloidaldispersion systems include, for example, macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Incertain embodiments, the preferred colloidal system of this disclosureis a liposome. Liposomes are artificial membrane vesicles which areuseful as delivery vehicles in vitro and in vivo. RNA, DNA, and intactvirions can be encapsulated within the aqueous interior and be deliveredto cells in a biologically active form [Fraley, et al. (1981) TrendsBiochem. Sci., 6:77]. Methods for efficient gene transfer using aliposome vehicle are known in the art [Mannino, et al. (1988)Biotechniques, 6:682, 1988].

The composition of the liposome is usually a combination ofphospholipids, which may include a steroid (e.g. cholesterol). Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations. Other phospholipids or other lipidsmay also be used including, for example a phosphatidyl compound (e.g.,phosphatidylglycerol, phosphatidylcholine, phosphatidylserine,phosphatidylethanolamine, a sphingolipid, a cerebroside, and aganglioside), egg phosphatidylcholine, dipalmitoylphosphatidylcholine,and distearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1. Generation of an ALK4:ActRIIB Heterodimer

Applicants constructed a soluble ALK4-Fc:ActRIIB-Fc heteromeric complexcomprising the extracellular domains of human ActRIIB and human ALK4,which are each separately fused to an Fc domain with a linker positionedbetween the extracellular domain and the Fc domain. The individualconstructs are referred to as ActRIIB-Fc fusion polypeptide and ALK4-Fcfusion polypeptide, respectively, and the sequences for each areprovided below.

A methodology for promoting formation of ALK4-Fc:ActRIIB-Fc heteromericcomplexes, as opposed to ActRIIB-Fc or ALK4-Fc homodimeric complexes, isto introduce alterations in the amino acid sequence of the Fc domains toguide the formation of asymmetric heteromeric complexes. Many differentapproaches to making asymmetric interaction pairs using Fc domains aredescribed in this disclosure.

In one approach, illustrated in the ActRIIB-Fc and ALK4-Fc polypeptidesequences of SEQ ID NOs: 39-41 and 42-44, respectively, one Fc domain isaltered to introduce cationic amino acids at the interaction face, whilethe other Fc domain is altered to introduce anionic amino acids at theinteraction face. ActRIIB-Fc fusion polypeptide and ALK4-Fc fusionpolypeptide each employ the tissue plasminogen activator (TPA) leader:MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 38).

The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 39) is shown below:

(SEQ ID NO: 39)  1 MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS 51 GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP251 APIEKTISKA KGQPREPQVY TLPPSRKEMT KNQVSLTCLV KGFYPSDIAV301 EWESNGQPEN NYKTTPPVLK SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH351 EALHNHYTQK SLSLSPGK

The leader (signal) sequence and linker are underlined. To promoteformation of ALK4-Fc:ActRIIB-Fc heterodimer rather than either of thepossible homodimeric complexes, two amino acid substitutions (replacingacidic amino acids with lysine) can be introduced into the Fc domain ofthe ActRIIB fusion protein as indicated by double underline above. Theamino acid sequence of SEQ ID NO: 39 may optionally be provided withlysine (K) removed from the C-terminus.

This ActRIIB-Fc fusion protein is encoded by the following nucleic acidsequence (SEQ ID NO: 40):

(SEQ ID NO: 40)   1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC  51 AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG 101 AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC 151 GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC 201 CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT 251 GGCTAGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG 301 GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA 351 GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC 401 CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC 451 CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA 501 ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG 551 TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG 601 GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA 651 CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT 701 GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA 751 GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC 801 ACAGGTGTAC ACCCTGCCCC CATCCCGGAA GGAGATGACC AAGAACCAGG 851 TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG 901 GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC 951 CGTGCTGAAG TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG 1101 TAAA

The mature ActRIIB-Fc fusion polypeptide (SEQ ID NO: 41) is as follows,and may optionally be provided with lysine (K) removed from theC-terminus.

(SEQ ID NO: 41)  1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT 51 IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS251 RKEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLKSDGSF301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

The complementary form of ALK4-Fc fusion polypeptide (SEQ ID NO: 42) isas follows:

(SEQ ID NO: 42)  1 MDAMKRGLCC VLLLCGAVFV SPGASGPRGV QALLCACTSC LQANYTCETD 51 GACMVSIFNL DGMEHHVRTC IPKVELVPAG KPFYCLSSED LRNTHCCYTD101 YCNRIDLRVP SGHLKEPEHP SMWGPVETGG GTHTCPPCPA PELLGGPSVF151 LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP201 REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG251 QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY301 DTTPPVLDSD GSFFLYSDLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL 351 SLSPG

The leader sequence and linker are underlined. To guide heterodimerformation with the ActRIIB-Fc fusion polypeptide of SEQ ID NOs: 39 and41 above, two amino acid substitutions (replacing lysines with asparticacids) can be introduced into the Fc domain of the ALK4-Fc fusionpolypeptide as indicated by double underline above. The amino acidsequence of SEQ ID NO: 42 may optionally be provided with lysine (K)added at the C-terminus.

This ALK4-Fc fusion protein is encoded by the following nucleic acid(SEQ ID NO: 43):

(SEQ ID NO: 43)   1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC  51 AGTCTTCGTT TCGCCCGGCG CCTCCGGGCC CCGGGGGGTC CAGGCTCTGC 101 TGTGTGCGTG CACCAGCTGC CTCCAGGCCA ACTACACGTG TGAGACAGAT 151 GGGGCCTGCA TGGTTTCCAT TTTCAATCTG GATGGGATGG AGCACCATGT 201 GCGCACCTGC ATCCCCAAAG TGGAGCTGGT CCCTGCCGGG AAGCCCTTCT 251 ACTGCCTGAG CTCGGAGGAC CTGCGCAACA CCCACTGCTG CTACACTGAC 301 TACTGCAACA GGATCGACTT GAGGGTGCCC AGTGGTCACC TCAAGGAGCC 351 TGAGCACCCG TCCATGTGGG GCCCGGTGGA GACCGGTGGT GGAACTCACA 401 CATGCCCACC GTGCCCAGCA CCTGAACTCC TGGGGGGACC GTCAGTCTTC 451 CTCTTCCCCC CAAAACCCAA GGACACCCTC ATGATCTCCC GGACCCCTGA 501 GGTCACATGC GTGGTGGTGG ACGTGAGCCA CGAAGACCCT GAGGTCAAGT 551 TCAACTGGTA CGTGGACGGC GTGGAGGTGC ATAATGCCAA GACAAAGCCG 601 CGGGAGGAGC AGTACAACAG CACGTACCGT GTGGTCAGCG TCCTCACCGT 651 CCTGCACCAG GACTGGCTGA ATGGCAAGGA GTACAAGTGC AAGGTCTCCA 701 ACAAAGCCCT CCCAGCCCCC ATCGAGAAAA CCATCTCCAA AGCCAAAGGG 751 CAGCCCCGAG AACCACAGGT GTACACCCTG CCCCCATCCC GGGAGGAGAT 801 GACCAAGAAC CAGGTCAGCC TGACCTGCCT GGTCACATGC TTCTATCCCA 851 GCGACATCGC CGTGGAGTGG GAGAGCAATG GGCAGCCGGA GAACAACTAC 901 GACACCACGC CTCCCGTGCT GGACTCCGAC GGCTCCTTCT TCCTCTATAG 951 CGACCTCACC GTGGACAAGA GCAGGTGGCA GCAGGGGAAC GTCTTCTCAT1001 GCTCCGTGAT GCATGAGGCT CTGCACAACC ACTACACGCA GAAGAGCCTC1051 TCCCTGTCTC CGGGT

The mature ALK4-Fc fusion protein sequence (SEQ ID NO: 44) is as followsand may optionally be provided with lysine (K) added at the C-terminus.

(SEQ ID NO: 44)  1 SGPRGVQALL CACTSCLQAN YTCETDGACM VSIFNLDGME HHVRTCIPKV 51 ELVPAGKPFY CLSSEDLRNT HCCYTDYCNR IDLRVPSGHL KEPEHPSMWG101 PVETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD151 VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN201 GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL251 TCLVKGFYPS DIAVEWESNG QPENNYDTTP PVLDSDGSFF LYSDLTVDKS301 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP G

The ActRIIB-Fc and ALK4-Fc proteins of SEQ ID NO: 41 and SEQ ID NO: 44,respectively, may be co-expressed and purified from a CHO cell line, togive rise to a heteromeric complex comprising ALK4-Fc:ActRIIB-Fc.

In another approach to promote the formation of heteromultimer complexesusing asymmetric Fc fusion proteins the Fc domains are altered tointroduce complementary hydrophobic interactions and an additionalintermolecular disulfide bond as illustrated in the ActRIIB-Fc andALK4-Fc polypeptide sequences of SEQ ID NOs: 45-46 and 47-48,respectively. The ActRIIB-Fc fusion polypeptide and ALK4-Fc fusionpolypeptide each employ the tissue plasminogen activator (TPA) leader:MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 38).

The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 45) is shown below:

(SEQ ID NO: 45)  1 MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS 51 GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP251 APIEKTISKA KGQPREPQVY TLPPCREEMT KNQVSLWCLV KGFYPSDIAV301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH351 EALHNHYTQK SLSLSPGK

The leader (signal) sequence and linker are underlined. To promoteformation of the ALK4-Fc:ActRIIB-Fc heterodimer rather than either ofthe possible homodimeric complexes, two amino acid substitutions(replacing a serine with a cysteine and a threonine with a trytophan)can be introduced into the Fc domain of the fusion protein as indicatedby double underline above. The amino acid sequence of SEQ ID NO: 45 mayoptionally be provided with lysine (K) removed from the C-terminus.

The mature ActRIIB-Fc fusion polypeptide is as follows:

(SEQ ID NO: 46)  1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT 51 IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPC251 REEMTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

The complementary form of ALK4-Fc fusion polypeptide (SEQ ID NO: 47) isas follows and may optionally be provided with lysine (K) removed fromthe C-terminus.

(SEQ ID NO: 47)  1 MDAMKRGLCC VLLLCGAVFV SPGASGPRGV QALLCACTSC LQANYTCETD 51 GACMVSIFNL DGMEHHVRTC IPKVELVPAG KPFYCLSSED LRNTHCCYTD101 YCNRIDLRVP SGHLKEPEHP SMWGPVETGG GTHTCPPCPA PELLGGPSVF151 LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP201 REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG251 QPREPQVCTL PPSREEMTKN QVSLSCAVKG FYPSDIAVEW ESNGQPENNY301 KTTPPVLDSD GSFFLVSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL 351 SLSPGK

The leader sequence and the linker are underlined. To guide heterodimerformation with the ActRIIB-Fc fusion polypeptide of SEQ ID NOs: 45 and46 above, four amino acid substitutions can be introduced into the Fcdomain of the ALK4 fusion polypeptide as indicated by double underlineabove. The amino acid sequence of SEQ ID NO: 47 may optionally beprovided with lysine (K) removed from the C-terminus.

The mature ALK4-Fc fusion protein sequence is as follows and mayoptionally be provided with lysine (K) removed from the C-terminus.

(SEQ ID NO: 48)  1 SGPRGVQALL CACTSCLQAN YTCETDGACM VSIFNLDGME HHVRTCIPKV 51 ELVPAGKPFY CLSSEDLRNT HCCYTDYCNR IDLRVPSGHL KEPEHPSMWG101 PVETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD151 VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN201 GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVCTLPPSR EEMTKNQVSL251 SCAVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LVSKLTVDKS301 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

ActRIIB-Fc and ALK4-Fc proteins of SEQ ID NO: 46 and SEQ ID NO: 48,respectively, may be co-expressed and purified from a CHO cell line, togive rise to a heteromeric complex comprising ALK4-Fc:ActRIIB-Fc.

Purification of various ALK4-Fc:ActRIIB-Fc complexes could be achievedby a series of column chromatography steps, including, for example,three or more of the following, in any order: protein A chromatography,Q sepharose chromatography, phenylsepharose chromatography, sizeexclusion chromatography, and cation exchange chromatography. Thepurification could be completed with viral filtration and bufferexchange.

Example 2. Ligand Binding Profile of ALK4-Fc:ActRIIB-Fc HeterodimerCompared to ActRIIB-Fc Homodimer and ALK4-Fc Homodimer

A Biacore™-based binding assay was used to compare ligand bindingselectivity of the ALK4-Fc:ActRIIB-Fc heterodimeric complex describedabove with that of ActRIIB-Fc and ALK4-Fc homodimer complexes. TheALK4-Fc: ActRIIB-Fc heterodimer, ActRIIB-Fc homodimer, and ALK4-Fchomodimer were independently captured onto the system using an anti-Fcantibody. Ligands were injected and allowed to flow over the capturedreceptor protein. Results are summarized in the table below, in whichligand off-rates (k_(d)) most indicative of effective ligand traps aredenoted in bold.

Ligand binding profile of ALK4-Fc:ActRIIB-Fc heterodimer compared toActRIIB-Fc homodimer and ALK4-Fc homodimer ActRIIB-Fc ALK4-FcALK4-Fc:ActRIIB-Fc homodimer homodimer heterodimer k_(a) k_(d) K_(D)k_(a) k_(d) K_(D) k_(a) k_(b) K_(D) Ligand (1/Ms) (1/s) (pM) (1/Ms)(1/s) (pM) (1/Ms) (1/s) (pM) Activin A 1.2 × 10⁷ 2.3 × 10 ⁻⁴ 19 5.8 ×10⁵ 1.2 × 10⁻²  20000 1.3 × 10⁷ 1.5 × 10 ⁻⁴ 12 Activin B 5.1 × 10⁶ 1.0 ×10 ⁻⁴ 20 No binding 7.1 × 10⁶ 4.0 × 10 ⁻⁵ 6 BMP6 3.2 × 10⁷ 6.8 × 10⁻³190 — 2.0 × 10⁶ 5.5 × 10⁻³ 2700 BMP9 1.4 × 10⁷ 1.1 × 10⁻³ 77 —Transient* 3400 BMP10 2.3 × 10⁷ 2.6 × 10 ⁻⁴ 11 — 5.6 × 10⁷ 4.1 × 10⁻³ 74GDF3 1.4 × 10⁶ 2.2 × 10⁻³ 1500 — 3.4 × 10⁶ 1.7 × 10⁻² 4900 GDF8 8.3 ×10⁵ 2.3 × 10 ⁻⁴ 280 1.3 × 10⁵ 1.9 × 10⁻³ 15000† 3.9 × 10⁵ 2.1 × 10 ⁻⁴550 GDF11 5.0 × 10⁷ 1.1 × 10 ⁻⁴ 2 5.0 × 10⁶ 4.8 × 10⁻³  270† 3.8 × 10⁷1.1 × 10 ⁻⁴ 3 *Indeterminate due to transient nature of interaction†Very low signal —Not tested

These comparative binding data demonstrate that ALK4-Fc:ActRIIB-Fcheterodimer has an altered binding profile/selectivity relative toeither ActRIIB-Fc or ALK4-Fc homodimers. ALK4-Fc:ActRIIB-Fc heterodimerdisplays enhanced binding to activin B compared with either homodimer,retains strong binding to activin A, GDF8, and GDF11 as observed withActRIIB-Fc homodimer, and exhibits substantially reduced binding toBMP9, BMP10, and GDF3. In particular, BMP9 displays low or no observableaffinity for ALK4-Fc:ActRIIB-Fc heterodimer, whereas this ligand bindsstrongly to ALK4-Fc:ActRIIB-Fc heterodimer. Like the ActRIIB-Fchomodimer, the heterodimer retains intermediate-level binding to BMP6.See FIG. 6.

In addition, an A-204 Reporter Gene Assay was used to evaluate theeffects of ALK4-Fc:ActRIIB-Fc heterodimer and ActRIIB-Fc:ActRIIB-Fchomodimer on signaling by activin A, activin B, GDF11, GDF8, BMP10, andBMP9. Cell line: Human Rhabdomyosarcoma (derived from muscle). Reportervector: pGL3(CAGA)12 (as described in Dennler et al, 1998, EMBO 17:3091-3100). The CAGA12 motif is present in TGF-beta responsive genes(PAI-1 gene), so this vector is of general use for factors signalingthrough Smad2 and 3. An exemplary A-204 Reporter Gene Assay is outlinedbelow.

-   -   Day 1: Split A-204 cells into 48-well plate.    -   Day 2: A-204 cells transfected with 10 ug pGL3(CAGA)12 or        pGL3(CAGA)12(10 ug)+pRLCMV (1 ug) and Fugene.    -   Day 3: Add factors (diluted into medium+0.1% BSA). Inhibitors        need to be pre-incubated with Factors for about one hr before        adding to cells. About six hrs later, cells are rinsed with PBS        and then lysed.

Following the above steps, applicant performed a Luciferase assay.

Both the ALK4-Fc:ActRIIB-Fc heterodimer and ActRIIB-Fc:ActRIIB-Fchomodimer were determined to be potent inhibitors of activin A, activinB, GDF11, and GDF8 in this assay. In particular, as can be seen in thecomparative homodimer/heterodimer IC₅₀ data illustrated in FIG. 13,ALK4-Fc:ActRIIB-Fc heterodimer inhibits activin A, activin B, GDF8, andGDF11 signaling pathways similarly to the ActRIIB-Fc:ActRIIB-Fchomodimer. However, ALK4-Fc:ActRIIB-Fc heterodimer inhibition of BMP9and BMP10 signaling pathways is significantly reduced compared to theActRIIB-Fc:ActRIIB-Fc homodimer. This data is consistent with theabove-discussed binding data in which it was observed that both theALK4-Fc:ActRIIB-Fc heterodimer and ActRIIB-Fc:ActRIIB-Fc homodimerdisplay strong binding to activin A, activin B, GDF8, and GDF11, butBMP10 and BMP9 have significantly reduced affinity for theALK4-Fc:ActRIIB-Fc heterodimer compared to the ActRIIB-Fc:ActRIIB-Fchomodimer.

Together, these data therefore demonstrate that ALK4-Fc:ActRIIB-Fcheterodimer is a more selective antagonist of activin B, activin A,GDF8, and GDF11 compared to ActRIIB-Fc homodimer. Accordingly, anALK4-Fc:ActRIIB-Fc heterodimer will be more useful than an ActRIIB-Fchomodimer in certain applications where such selective antagonism isadvantageous. Examples include therapeutic applications where it isdesirable to retain antagonism of one or more of activin A, activin B,activin AB, GDF8, and GDF11 but minimize antagonism of one or more ofBMP9, BMP10, GDF3, and BMP6.

Example 3. Activity Profile of ALK4-Fc:ActRIIB-Fc Heterodimer in MiceCompared to ActRIIB-Fc Homodimer

Homodimeric and heterodimeric complexes were tested in mice toinvestigate differences in their activity profiles in vivo. Wild-typeC57BL/6 mice were dosed subcutaneously with ActRIIB-Fc homodimer (10mg/kg), ALK4-Fc:ActRIIB-Fc heterodimer (3 or 10 mg/kg), or vehicle(phosphate-buffered saline, PBS) twice per week for 4 weeks beginning atapproximately 10 weeks of age (n=9 mice per group). ALK4-Fc homodimerwas not tested in vivo due to its inability to bind ligands with highaffinity under cell-free conditions as determined by surface plasmonresonance. Study endpoints included: body weight; total lean mass andtotal adipose mass as determined by nuclear magnetic resonance (NMR) atbaseline and study completion (4 weeks); total bone mineral density asdetermined by dual energy x-ray absorptiometry (DEXA) at baseline and 4weeks; and weights of the gastrocnemius, rectus femoris, and pectoralismuscles determined at 4 weeks.

Activity of ActRIIB-Fc and ALK4-Fc Complexes in Wild-Type MiceActRIIB-Fc ALK4-Fc:ActRIIB-Fc Endpoint homodimer heterodimer (4 wk)Vehicle 10 mg/kg 10 mg/kg 3 mg/kg Change in body weight ↑ 15%  ↑ 38%** ↑41%** ↑ 33%** from baseline Change in total lean mass ↓ 1%  ↑ 5%**  ↑5%**  ↑ 5%** from baseline Change in total adipose ↑ 5% ↓ 3.6%**  ↓3.5%**  ↓ 3.5%**  mass from baseline Change in total bone ↑ 8% ↑ 14%*  ↑12%*  ↑ 11%  mineral density from baseline Gastrocnemius weight † 2336** 35** 30** Femoris weight † 11.5 17** 16** 14** Pectoralis weight †15 23** 28** 23** *P < 0.05 vs. vehicle **P < 0.01 vs. vehicle †Combined left and right muscle weights normalized to femur length(mg/mm) to control for body size

Study results are summarized in the table above. As expected, ActRIIB-Fchomodimer caused marked changes in body composition, many consistentwith known effects of GDF8 and activin inhibition. Treatment ofwild-type mice with ActRIIB-Fc homodimer more than doubled body weightgain over the course of the study compared to vehicle-treated controls.Accompanying this net weight gain were significant increases in totallean mass and total bone mineral density, as well as a significantreduction in total adipose mass, compared to vehicle. It should berecognized that normalized (percentage-based) changes in lean andadipose tissues differ in their correspondence to absolute changesbecause lean mass (typically about 70% of body weight in a mouse) ismuch larger than adipose mass (typically about 10% of body weight).Individual skeletal muscles examined, including the gastrocnemius,femoris, and pectoralis all increased significantly in weight comparedto vehicle controls over the course of treatment with ActRIIB-Fchomodimer.

The ALK4-Fc:ActRIIB-Fc heterodimer produced certain effects strikinglysimilar to those of the ActRIIB-Fc homodimer despite differential ligandselectivity of the two complexes. As shown in the table above, treatmentof mice with the ALK4-Fc:ActRIIB-Fc heterodimer at a dose level of 10mg/kg matched, nearly matched, or exceeded the effects of ActRIIB-Fchomodimer at the same dose level for all endpoints listed. Effects ofthe ALK4-Fc:ActRIIB-Fc heterodimer at 3 mg/kg were mildly attenuated forseveral endpoints compared to 10 mg/kg, thus providing evidence for adose-effect relationship.

Thus, an ALK4-Fc:ActRIIB-Fc heterodimer exerts beneficial anaboliceffects on skeletal muscle and bone, and catabolic effects on adiposetissue, very similar to those of ActRIIB-Fc homodimer. However, unlikeActRIIB homodimer, ALK4-Fc:ActRIIB-Fc heterodimer exhibits onlylow-affinity or transient binding to BMP9 and BMP10 and so should havelittle to no concurrent inhibition on processes mediated by thoseligands, such as angiogenesis. This novel selectivity will be useful,for example, in treating patients in need of stimulatory effects onmuscle and bone, and inhibitory effects on fat, but not in need ofaltered angiogenesis.

Example 4. ALK4:ActRIIB Heteromultimer Treatment Suppresses KidneyFibrosis and Inflammation and Reduces Kidney Injury

The effects of the ALK4-Fc:ActRIIB-Fc heterodimer described in Example 2on kidney disease was assessed in a mouse unilateral ureteralobstruction model. See, e.g., Klahr and Morrissey (2002) Am J PhysiolRenal Physiol 283: F861-F875.

Twenty-four C57BL/6 male mice 12 weeks of age underwent left unilateralureteral ligation twice at the level of the lower pole of kidney. After3 days, eight mice were euthanized and kidneys from individual animalswere harvested to assess kidney injury. The remaining mice wererandomized into two groups: i) eight mice were injected subcutaneouslywith the ALK4-Fc:ActRIIB-Fc heterodimer at a dose of 10 mg/kg at day 3,day 7, day 10, and day 14 after surgery and a ii) eight mice wereinjected subcutaneously with vehicle control, phosphate buffered saline(PBS), at day 3, day 7, day 10, and day 14 after surgery. Both groupswere sacrificed at day 17 in accordance with the relevant Animal CareGuidelines. Half kidneys from individual animals were collected forhistology analysis (H&E, and Masson's Trichrome stain), from both theUUO kidney and contralateral kidney, and 1/4 kidneys were used for RNAextraction (RNeasy Midi Kit, Qiagen, Ill.).

Gene expression analysis on UUO kidney samples was performed to assesslevels of various genes. QRT-PCR was performed on a CFX Connect™Real-time PCR detection system (Bio-Rad, CA) to evaluate the expressionof various fibrotic genes (Col1a1, Fibronectin, PAI-1, CTGF, and a-SMA),inflammatory genes (TNFa, and MCP1), cytokines (TGFβ1, TGFβ2, TGFβ3, andactivin A), and kidney injury genes (NGAL. See FIG. 14. Treatment ofmice with ALK4-Fc:ActRIIB-Fc heterodimer significantly suppressed theexpression of fibrotic and inflammatory genes, inhibited theupregulation of TGFβ 1/2/3 and reduced kidney injury. Histology dataconfirmed that ALK4-Fc:ActRIIB-Fc heterodimer treatment significantlyinhibited kidney fibrosis and reduced kidney injury in the UUO model.

Together, these data demonstrate that ALK4:ActRIIB heteromultimertreatment suppresses kidney fibrosis and inflammation and reduces kidneyinjury. Moreover, these data indicate that other ALK4:ActRIIBantagonists may be useful in the treatment or preventing of kidneydisease including, for example, antagonists of ALK4 and/orActRIIB-binding ligands, antagonists of ALK4 and/or ActRIIB receptors,antagonists of ALK4 and/or ActRIIB downstream signaling mediators (e.g.,Smads), and antagonists of TGFβ superfamily co-receptors associated withALK4 and/or ActRIIB.

We claim:
 1. A soluble recombinant heteromultimer comprising an ALK4polypeptide and an ActRIIB polypeptide, wherein the ALK4 polypeptidecomprises an amino acid sequence that is at least 90% identical to aminoacids 34-101 of SEQ ID NO: 9, and wherein the ActRIIB polypeptidecomprises an amino acid sequence that is at least 90% identical to aminoacids 29-109 of SEQ ID NO: 1, wherein the ActRIIB polypeptide does notcomprise an aspartic acid (D) at the amino acid position correspondingto L79 of SEQ ID NO: 1; wherein the ALK4 and/or ActRIIB polypeptide is afusion protein further comprising a heterologous domain, and wherein theheteromultimer binds to activin B.
 2. The heteromultimer of claim 1,wherein the ALK4 polypeptide comprises an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a polypeptide that: a) begins at any one of amino acids 24,25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 of SEQ ID NO: 9, and b) endsat any one of amino acids 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, or 126 of SEQ ID NO:
 9. 3. The heteromultimer of claim 1,wherein the ActRIIB polypeptide comprises an amino acid sequence that isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a polypeptide that: a) begins at any one of amino acids 20,21, 22, 23, 24, 25, 26, 27, 28, or 29 of SEQ ID NO: 1, and b) ends atany one of amino acids 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, or 134 of SEQ ID NO:
 1. 4. The heteromultimer of claim 1, whereinthe heterologous domain comprises an Fc immunoglobulin domain.
 5. Theheteromultimer of claim 4, wherein the Fc immunoglobulin domaincomprises one or more amino acid modifications that promote heterodimerformation.
 6. The heteromultimer of claim 4, wherein the immunoglobulinFc domain comprises one or more amino acid modifications that inhibithomodimer formation.
 7. The heteromultimer of claim 4, wherein theheterologous domain comprises an Fc immunoglobulin domain of IgG1immunoglobulin.
 8. The heteromultimer of claim 1, wherein the fusionprotein further comprises a linker domain positioned between the ALK4domain and the heterologous domain and/or a linker domain positionedbetween the ActRIIIB domain and the heterologous domain.
 9. Theheteromultimer of claim 1, wherein the ALK4 polypeptide and/or ActRIIBpolypeptide comprises one or more modified amino acid residues selectedfrom the group consisting of: a glycosylated amino acid, a PEGylatedamino acid, a farnesylated amino acid, an acetylated amino acid, abiotinylated amino acid, and an amino acid conjugated to a lipid moiety.10. The heteromultimer of claim 1, wherein the ALK4 polypeptide and/orActRIIB polypeptide is glycosylated and has a glycosylation patternobtainable from a Chinese hamster ovary cell line.
 11. Theheteromultimer of claim 1, wherein the heteromultimer is an ALK4:ActRIIBheterodimer.
 12. A pharmaceutical preparation comprising theheteromultimer of claim 1 and a pharmaceutically acceptable carrier. 13.The pharmaceutical preparation of claim 12, wherein the pharmaceuticalpreparation is substantially pyrogen-free.
 14. The pharmaceuticalpreparation of claim 12, wherein the pharmaceutical preparation furthercomprises an additional active agent.
 15. The pharmaceutical preparationof claim 12, wherein the pharmaceutical preparation comprises less than10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% ALK4 homomultimers.16. The pharmaceutical preparation of claim 12, wherein thepharmaceutical preparation comprises less than 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, or less than 1% ActRIIB homomultimers.
 17. Theheteromultimer of claim 1, wherein the ALK4 polypeptide comprises anamino acid sequence that is at least 95% identical to amino acids 34-101of SEQ ID NO: 9, and wherein the ActRIIB polypeptide comprises an aminoacid sequence that is at least 95% identical to amino acids 29-109 ofSEQ ID NO:
 1. 18. The heteromultimer of claim 1, wherein the ALK4polypeptide comprises the amino acid sequence corresponding to aminoacids 34-101 of SEQ ID NO: 9, and wherein the ActRIIB polypeptidecomprises the amino acid sequence corresponding to amino acids 29-109 ofSEQ ID NO:
 1. 19. The heteromultimer of claim 1, wherein the ALK4polypeptide comprises an amino acid sequence that is at least 90%identical to the amino acid sequence of SEQ ID NO: 10, and wherein theActRIIB polypeptide comprises an amino acid sequence that is at least90% identical to the amino acid sequence of SEQ ID NO:
 2. 20. Theheteromultimer of claim 1, wherein the ALK4 polypeptide comprises theamino acid sequence of SEQ ID NO: 10, and wherein the ActRIIBpolypeptide comprises the amino acid sequence of SEQ ID NO:
 2. 21. Theheteromultimer of claim 17, wherein the heterologous domain comprises anFc immunoglobulin domain, wherein the Fc immunoglobulin domain is anIgG1 immunoglobulin domain.
 22. The heteromultimer of claim 18, whereinthe heterologous domain comprises an Fc immunoglobulin domain, whereinthe Fc immunoglobulin domain is an IgG1 immunoglobulin domain.
 23. Theheteromultimer of claim 19, wherein the heterologous domain comprises anFc immunoglobulin domain, wherein the Fc immunoglobulin domain is anIgG1 immunoglobulin domain.
 24. The heteromultimer of claim 20, whereinthe heterologous domain comprises an Fc immunoglobulin domain, whereinthe Fc immunoglobulin domain is an IgG1 immunoglobulin domain.
 25. Theheteromultimer of claim 17, wherein the fusion protein further comprisesa linker domain positioned between the ALK4 domain and the heterologousdomain and/or a linker domain positioned between the ActRIIB domain andthe heterologous domain.
 26. The heteromultimer of claim 18, wherein thefusion protein further comprises a linker domain positioned between theALK4 domain and the heterologous domain and/or a linker domainpositioned between the ActRIIB domain and the heterologous domain. 27.The heteromultimer of claim 19, wherein the fusion protein furthercomprises a linker domain positioned between the ALK4 domain and theheterologous domain and/or a linker domain positioned between theActRIIB domain and the heterologous domain.
 28. The heteromultimer ofclaim 20, wherein the fusion protein further comprises a linker domainpositioned between the ALK4 domain and the heterologous domain and/or alinker domain positioned between the ActRIIB domain and the heterologousdomain.
 29. The heteromultimer of claim 1, wherein the ALK4 polypeptidecomprises an amino acid sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO: 10, and wherein the ActRIIBpolypeptide comprises an amino acid sequence that is at least 95%identical to the amino acid sequence of SEQ ID NO:
 2. 30. Theheteromultimer of claim 29, wherein the heterologous domain comprises anFc immunoglobulin domain, wherein the Fc immunoglobulin domain is anIgG1 immunoglobulin domain.
 31. The heteromultimer of claim 29, whereinthe fusion protein further comprises a linker domain positioned betweenthe ALK4 domain and the heterologous domain and/or a linker domainpositioned between the ActRIIB domain and the heterologous domain.